Peptides derived from fibronectin with improved bioactivity and reduced susceptibility to neutrophil elastase degradation

ABSTRACT

Polypeptides derived from fibronectin are presented that are neutrophil elastase-resistant and can bind to growth factors and/or enhance growth factor activity. These polypeptides are useful for enhancing wound healing in a patient.

GOVERNMENT SUPPORT

This invention was made with government support awarded by US Army AFIRM1 and JWMRP S14 under grant numbers W81XWH-08-2-0034 andW81XWH-15-C-0043, respectively. The government has certain rights in theinvention.

STATEMENT REGARDING ELECTRONIC FILING OF A SEQUENCE LISTING

A Sequence Listing in XML format, entitled 1543-3CT_070623_ST26.xml,149,483 bytes in size, generated on Jul. 6, 2023, and filed herewith, ishereby incorporated by reference in its entirety for its disclosures.

TECHNICAL FIELD

This invention is based on the discovery of polypeptides derived fromfibronectin that are neutrophil elastase-resistant and can bind togrowth factors and/or enhance growth factor activity. Previously knownpolypeptides derived from fibronectin with the ability to enhance growthfactor activity are found to be susceptible to degradation by humanneutrophil elastase, reducing their effectiveness in in vivo uses. Thisinvention relates to novel growth factor enhancing polypeptides withincreased resistance to degradation by neutrophil elastase. Thisinvention also relates to the uses of such compounds in cosmetictreatments and the treatment of wounds and cancer.

BACKGROUND

In the US civilian population, each year, approximately 500,000 patientswith burns present to emergency departments. Of 40,000 annual hospitaladmissions, 25,000 burn victims are admitted to specialized burncenters. Progressive extension of burns can have a devastating effect.Over the course of a few days to one week deep partial-thickness burnscan become full-thickness burns, which in the short term, leads toincreased tissue loss, longer healing time, excess morbidity andmortality. In the long term, increased scarring, wound contractures andpoor quality of life become major issues. While the exact mechanism(s)leading to conversion of the zone of ischemia to full-blown necrosis isunclear, several processes, including oxidant and cytokine stressresulting from inflammation as well as ischemia/reperfusion, probablyplay a role.

Therapies to improve blood flow, such as non-steroidal anti-inflammatoryagents (NSAIDS) and anti-coagulants (heparin) have not shown substantialbenefit in preventing burn injury progression. Hence therapy to limitburn injury progression is an unmet need.

There is evidence that fibronectin (FN) is involved in many biologicalprocesses including tissue repair, embryogenesis, blood clotting, cellmigration, wound repair, and cell adhesion. There are two primary formsof fibronectin. The first is an insoluble glycoprotein dimer that servesas a linker in the extracellular matrix (ECM), and the second is asoluble disulfide-linked dimer found in plasma. The ECM form offibronectin is expressed by fibroblasts, chondrocytes, endothelialcells, macrophages and certain epithelial cells. The plasma form offibronectin is expressed by hepatocytes. Fibronectin can serve as ageneral cell adhesion molecule by anchoring cells to collagen or toproteoglycan substrates. Fibronectin can also play a role in organizingcellular interactions by binding to components of the ECM and tomembrane-bound fibronectin receptors on cell surfaces. Forms offibronectin are found in vertebrates, including mammals, birds,amphibians, fish, and reptiles.

FN, a 500 kDa glycoprotein, circulates in the blood and is produced anddeposited by tissue cells in the provisional extracellular matrix (ECM)during tissue formation. As a critical component of the provisional ECM,FN plays a vital role in embryogenesis, morphogenesis and wound healingbut is deficient in burn patients' wounds and blood. FN is known to bedegraded in burn wound fluids by the endopeptidase neutrophil elastase.See Grinnell, et al., Identification of Neutrophil Elastase as theProteinase in Burn Wound Fluid Responsible for Degradation ofFibronectin, J Invest. Dermatology, 1994, 103(2):155-61.

Previously disclosed peptide “P12” is 14-residue peptide that is crypticwithin the immunoglobulin sandwich type of β-pleated sheet offibronectin's (FN) first type III repeat (FNIII₁). Unlike FN, P12 insolution promotes mesenchymal cell growth, proliferation and migrationintrinsically and synergistically with a variety of growth factors,especially platelet-derived growth factor-BB (PDGF-BB). Furthermore, P12protects adult human dermal fibroblasts (AHDF) from cell death inducedby oxidative and cytokine stress and/or nutrient withdrawal in thepresence of PDGF-BB. P12 also limits burn injury progression in rat andporcine burn models and mitigates scarring in a vertical burn injuryprogression pig model. See, e.g., PCT/US2006/038778; U.S. Pat. No.8,759,300; Lin, et al., Fibronectin peptides that bind PDGF-BB enhancesurvival of cells and tissue under stress, J Invest Dermatol. 2014April; 134(4): 1119-1127; and Asif, et al., Blood Vessel Occlusion inPeri-burn Tissue is Secondary to Erythrocyte Aggregation and Mitigatedby a Fibronectin-derived Peptide that Limits Burn Injury Progression,Wound Rep Reg (2016) 24 501-513. In particular, the fragment offibronectin PSHISKYILRWRPK (SEQ ID NO:108), or “P12” is disclosed inboth Lin, et al. and Asif, et al. as being useful for the treatment ofwounds, particularly the treatment of burns.

SUMMARY

We present neutrophil elastate-resistant peptides derived from fragmentsof fibronectin for use in the treatment of wounds. In particular, wehave discovered that previously disclosed, biologically active, peptidefragments of fibronectin, such as P12, are readily degraded, in vitro,by neutrophil elastase, and do not promote healing when appliedtopically to wounds. Even cyclized forms of the previously disclosed,biologically active, peptide fragments of fibronectin, such as cyclicP12, remain sensitive to this endopeptidase. We present peptides thatare fragments and/or derivatives of fragments of fibronectin thatmaintain their biological activity of binding growth factors, such asplatelet-derived growth factor-BB (PDGF-BB) and enhancing fibroblastsurvival, and also have been modified to increase their resistance todegradation by neutrophil elastase, in vitro and in vivo.

In some embodiments of the invention, the neutrophil elastase resistantpeptide is a linear or cyclic polypeptide according to formula I:

(SEQ ID NO: 1) (I): H-X₁-X₂-K-Y-X₃-X₄-R-W-R-P-K-X₅-X₆-X₇

-   -   wherein X₁ is I or G or L,        -   X₂ is S or G,        -   X₃ is I or G or L,        -   X₄ is L or G,        -   X₅ is N or G,        -   X₆ is absent or S, and        -   X₇ is absent or V; and

wherein no two consecutive amino acids in the first 13 amino acids ofthe polypeptide differ from the sequence HISKYILRWRPKN (SEQ ID NO:9). Anembodiment of this invention wherein X₆ of formula I is absent is setforth in the Sequence Listing as SEQ ID NO:2. An embodiment of thisinvention wherein X₆ and X₇ of formula I are absent is set forth in theSequence Listing as SEQ ID NO:3. An embodiment of this invention whereinX₇ of formula I is absent is set forth in the Sequence Listing as SEQ IDNO:4.

Further embodiments of the invention include HISKYILRWRPKNSV (SEQ IDNO:10) (P46), HIGKYGLRWRPKNSV (SEQ ID NO:11) (cNP7), HIGKYGLRWRPKGSV(SEQ ID NO:12) (cNP8), HGSKYGLRWRPKNSV (SEQ ID NO:13), HIGKYIGRWRPKNSV(SEQ ID NO:14), HGSKYIGRWRPKNSV (SEQ ID NO:15), HGSKYIGRWRPKGSV (SEQ IDNO:16) and cyclic forms thereof.

We also present methods of using these peptides for cosmetic treatmentsand the treatment of wounds and cardiovascular disease. The wounds to betreated include surgical incision or extirpation, a traumatic injury, athermal burn, a chemical burn, a lesion or ulceration of the patient'sskin, mucosa, connective tissue, fascia, ligament, tendon, cartilage,nerve or muscle and a wound to the patient's bone. The treated woundsmay be infected or uninfected. The cardiovascular incidents to betreated include blood vessels occluded with aggregates of red blood celland/or fibrinogen and/or fibrin, such as can occur in burn wounds;myocardial infarction; multi-organ failure; diabetes; sickle cellanemia; polycythemia vera; and hyperfibrinogenemia. In a particularembodiment, the neutrophil elastase resistant peptides of the inventionare used to treat thermal and/or chemical burns.

The invention also features compositions (e.g., physiologicallyacceptable compositions) that include a neutrophil elastase resistantpeptide of the invention. The physiologically acceptable composition maybe a pharmaceutical composition that promotes a therapeutic response. Asnoted above, cosmetic compositions are also featured and can include thepeptides described herein. The present compositions may also benon-pharmaceutical in the sense that they may include concentratedpeptides and/or other ingredients that should be diluted or otherwisemodified (e.g., mixed with other active or inactive ingredients) priorto use (e.g., in cell culture or as a cosmetic or therapeuticformulation).

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B show a diagram of the susceptibility of cP12 to digestion byhuman neutrophil elastase. FIG. 1A: cP12 was incubated with purifiedhuman neutrophil elastase at 1:200 enzyme:substrate molar ratio, minimalintact peptide (MW=1789) was retained at 24 h. FIG. 1B: Elastasesensitive sites of cP12 cleaved by human neutrophil elastase andanalyzed by MS. PSHISKYILRWRPK is SEQ ID NO:108; HISKYILPWRPKPS is SEQID NO:109; KYILRWRPKPSHIS is SEQ ID NO:110; LRWRKPSHISKYI is SEQ IDNO:111; and RWRPKPSHISKYIL is SEQ ID NO:112.

FIG. 2 shows bioactivity for P12, P45 and P46. Adult human dermalfibroblasts CF31 cells at 1000 cells/well were seeded in acollagen-coated 96-well plate in DMEM overnight, then 1nM PDGF-BB withpeptides at indicated doses was added and cells were incubated at 37° C.for 6 days. Then cell metabolism was measured by XTT assay by reading ODat 450 nm. Each point represents the mean of 6 replicates with a typicalSD=5-10% around the mean (not shown for graft clarity).

FIGS. 3A-3B show P46 digestion by neutrophil elastase at 4 hours and 24hours. P46 was incubated with purified human neutrophil elastase at1:100 enzyme:substrate molar ratio at 37° C. for 4 h (3A) or 24 h (3B).Peptides stability was determined by MALDI-TOF analysis. Molecularweight of P46 is 1898.

FIG. 4 shows bioactivity of P12, P46, cNP7 and cNP8. Adult human dermalfibroblasts, CF31, at 1000 cells/well were incubated at 37° C. in acollagen-coated 96-well plate with DMEM, 1nM PDGF-BB with peptides atindicated doses for 6 days and then cell metabolism was measured by XTTassay, reading OD at 450 nm. Data represent the mean of 6 replicates.

FIGS. 5A-5B show that cNP8, derived from P46, is resistant to digestionby neutrophil elastase. cNP8 was incubated with purified humanneutrophil elastase at 1:100 enzyme:substrate molar ratio at 37° C. for4 h (5A) or 24 h (5B). Peptides' stability was determined by MALDI-TOFanalysis. Molecular weight of cNP8 is 1736. Intact peptide (MW=1736) wasretained at 24 h.

FIG. 6 shows a comparison of effectiveness of cP12 and cNP8 peptide forpromoting re-epithelialization of burn wounds with topical treatment ina porcine model.

FIG. 7 shows the increase of re-epithelialization on day 10 at the woundsite with intravenous cNP8 treatment in a porcine burn model.

FIG. 8 shows the increase of re-epithelialization on day 14 at the woundsite with intravenous cNP8 treatment in a porcine burn model.

DETAILED DESCRIPTION

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

Determination of peptides derived from fibronectin with improvedbioactivity and resistance to neutrophil elastase digestion:

Previously disclosed peptide P12 is degraded by neutrophil elastase,which is present in wound fluids:

To determine whether cyclic P12 (“cP12”) is sensitive to endopeptidases,cP12 was digested with human neutrophil elastase. The reaction wasstopped with formic acid and the reaction mixture was analyzed by massspectroscopy. Analysis of results showed that cP12 has fiveelastase-cleavage sites (FIG. 1B). In addition, almost all intact cP12substrate (1789 mw) was eliminated (FIG. 1A).

Determination of fragments created by digestion of fibronectin withhuman neutrophil elastase, and their bioactivity:

To find an elastase-resistant cP12-derivative with cP12-equivalentbioactivity, we digested fibronectin III₁-C (FNIII₁-C), in which P12peptide is found, with purified human neutrophil elastase at 1:100enzyme:substrate molar ratio at 37° C. for 4 h or 24 h. Digested sampleswere analyzed with MS. The results at both time points showed thatelastase digestion of FN III₁-C produced two peptides, P45(SKYILRWRPKNSV) (SEQ ID NO:5) and P46 (HISKYILRWRPKNSV) (SEQ ID NO:10)from the same region of fibronectin as P12. Bioactivity assaydemonstrated that P46 showed higher bioactivity than P12 as determinedby fibroblast metabolism assay (FIG. 2 ). On the other hand, P45 showedlittle bioactivity (FIG. 2 ).

Determination of sensitivity of P46 to human neutrophil elastasedigestion:

To determine the sensitivity of P46 to elastase digestion, P46 wassynthesized and digested with elastase. The MS analysis results showedthat P46 retained some sensitivity to elastase digestion (FIGS. 3A-3B).P46 was incubated with purified human neutrophil elastase at 1:100enzyme:substrate molar ratio at 37° C. for 4 h (FIG. 3A) or 24 h (FIG.3B). The elastase-generated smaller peptides were determined byMALDI-TOF analysis. Molecular weight of P46 is 1898.

cNP7 and cNP8 as biologically active, neutrophil elastase-resistantpeptides:

Based on the amino acid sequence of P46 and enzymatic cleavageproperties of elastase, we designed and synthesized five engineeredpeptides, -HIGKYGLRWRPKNSV (SEQ ID NO:11)-(NP7) and -HIGKYGLRWRPKGSV(SEQ ID NO:12)-(NP8), HISKYILGWRPKNSV (SEQ ID NO:6) (NP9),HISKYILRGRPKNSV (SEQ ID NO:7) (NP10), HISKYILRWGPKNSV (SEQ ID NO:8)(NP11), and screened for resistance to elastase digestion. Of these, NP8demonstrated the best biological activity on screening experiment ofadult human dermal fibroblast survival in medium without serum but with1nM PDGF-BB after 6 days of incubation. NP9, NP10 and NP11 showedminimal biological activity in this screen.

P12, P46, cNP7 and cNP8 were compared for their ability to promote thesurvival of adult human dermal fibroblasts in the presence of PDGF-BB.Adult human dermal fibroblasts at 1000 cells/well were incubated in acollagen-coated 96-well plate with DMEM, 1 nM PDGF-BB with peptides atindicated doses for 6 days and then cell metabolism was measured by XTTassay. Data represent the mean of 6 replicates (FIG. 4 ). Thisbioactivity assay demonstrated all four peptides promoted the survivalof adult human dermal fibroblasts and that cNP8 showed equivalent orbetter bioactivity, depending on the concentration, compared to P46 andcNP7.

Results from peptide incubation with elastase showed that cNP8 wasresistant to elastase digestion. cNP8, derived from P46, was incubatedwith purified human neutrophil elastase at 1:100 enzyme:substrate molarratio at 37° C. for 4 h (FIG. 5A) or 24 h (FIG. 5B). Peptide's stabilitywas determined by MALDI-TOF analysis. Molecular weight of cNP8 is 1736.A large majority of intact peptide was retained at 24 h. Thus, cNP8 is apeptide with both bioactivity and resistant to elastase digestion.

p46 and cNP8 engineered from P46 have the ability to bind growthfactors:

Previous studies showed that fibronectin-derived bioactive peptide P12interacts with platelet-derived growth factor-BB and enhances itsactivity to support fibroblasts survival. We showed, above, that bothfibronectin-derived peptide P46 and elastase-resistant engineeredpeptide cNP8 demonstrated much higher bioactivity than that of P12. Tostudy the binding activities of these peptides, real time interactionsof P12, P46, and cNP8 with growth factors were determined by plasmonsurface resonance (Biacore T200). The results demonstrated that both P46and cNP8 showed similar binding activity as P12. They bind PDGF-BB andTransforming Growth Factor-Beta1 (TGF-β1), but did not bind to epidermalgrowth factor (EGF) and insulin-like growth factor-1 (IGF-1).

Surface Plasmon Resonance:

In the Biacore2000 system, binding constants from kinetic data aredetermined by passing varying concentrations of FN peptides (analytes)over chip surfaces which are coupled with PDGF-BB (ligand),respectively. All kinetic experiments are carried out at 20° C. at aflow rate of 20 μl/min For mass transport experiments, each analyte isinjected at a fixed concentration and run at flow rates ranging from 5to 75 μl/min. All analytes are injected over PDGF-BB ligand surfaces aswell as over a control surface for 120 s, followed by 300 s ofdissociation in running buffer. Regeneration of the sensor chip forsubsequent injections is accomplished by pulse of 0.1% SDS. Masstransport experiments have detected little difference in response atdifferent flow rates thus validating data from kinetic experiments.Sensorgrams are prepared and globally fitted using nonlinearleast-squares analysis and numerical integration of the differentialrate equations with the Biacore Bioevaluation software. Each sensorgramgenerated using a control surface is subtracted from the correspondingexperimental sensorgrams, and the resulting curves are transformed toconcentration units using the molecular mass of the injected species,the equivalence of 1000 resonance units (RU) per 1 ng/mm2, and a matrixthickness of 100 nm. Each data set, which consists of a series ofsensorgrams from injections of different concentration of analyte overthe same surface, is analyzed using kinetic models from Bioevaluationsoftware.

Novel peptides for use in wound healing:

Linear or cyclic peptides according to Formula I:

(SEQ ID NO: 1) (I): H-X₁-X₂-K-Y-X₃-X₄-R-W-R-P-K-X₅-X₆-X₇

-   -   wherein X₁ is I or G or L,        -   X₂ is S or G,        -   X₃ is I or G or L,        -   X₄is L or G,        -   X₅ is N or G,        -   X₆ is absent or S, and        -   X₇ is absent or V; and

wherein no two consecutive amino acids in the first 13 amino acids ofthe polypeptide differ from the sequence HISKYILRWRPKN (SEQ ID NO:9),are useful for the treatment of wounds. These peptides promote thesurvival, migration or growth of human adult dermal fibroblasts andhuman adult cardiomyocytes, and are resistant to neutrophil elastase, anendopeptidase found in wound fluids.

Each linear or cyclic peptide selected from: HISKYILRWRPKNSV (SEQ IDNO:10), HISKYILRWRPKNS (SEQ ID NO:17), HISKYILRWRPKN (SEQ ID NO:9),HISKYILRWRPKGSV (SEQ ID NO:18), HISKYILRWRPKGS (SEQ ID NO:19),HISKYILRWRPKG (SEQ ID NO:20), HGSKYILRWRPKNSV (SEQ ID NO:21),HGSKYILRWRPKNS (SEQ ID NO:22), HGSKYILRWRPKN (SEQ ID NO:23),HGSKYILRWRPKGSV (SEQ ID NO:24), HGSKYILRWRPKGS (SEQ ID NO:25),HGSKYILRWRPKG (SEQ ID NO:26), HLSKYILRWRPKNSV (SEQ ID NO:27),HLSKYILRWRPKNS (SEQ ID NO:28), HLSKYILRWRPKN (SEQ ID NO:29),HLSKYILRWRPKGSV (SEQ ID NO:30), HLSKYILRWRPKGS (SEQ ID NO:31),HLSKYILRWRPKG (SEQ ID NO:32), HIGKYILRWRPKNSV (SEQ ID NO:33),HIGKYILRWRPKNS (SEQ ID NO:34), HIGKYILRWRPKN (SEQ ID NO:35),HIGKYILRWRPKGSV (SEQ ID NO:36), HIGKYILRWRPKGS (SEQ ID NO:37),HIGKYILRWRPKG (SEQ ID NO:38), HISKYGLRWRPKNSV (SEQ ID NO:39),HISKYGLRWRPKNS (SEQ ID NO:40), HISKYGLRWRPKN (SEQ ID NO:41),HISKYGLRWRPKGSV (SEQ ID NO:42), HISKYGLRWRPKGS (SEQ ID NO:43),HISKYGLRWRPKG (SEQ ID NO:44), HGSKYGLRWRPKNSV (SEQ ID NO:13),HGSKYGLRWRPKNS (SEQ ID NO:45), HGSKYGLRWRPKN (SEQ ID NO:46),HGSKYGLRWRPKGSV (SEQ ID NO:47), HGSKYGLRWRPKGS (SEQ ID NO:48),HGSKYGLRWRPKG (SEQ ID NO:49), HLSKYGLRWRPKNSV (SEQ ID NO:50),HLSKYGLRWRPKNS (SEQ ID NO:51), HLSKYGLRWRPKN (SEQ ID NO:52),HLSKYGLRWRPKGSV (SEQ ID NO:53), HLSKYGLRWRPKGS (SEQ ID NO:54),HLSKYGLRWRPKG (SEQ ID NO:55), HIGKYGLRWRPKNSV (SEQ ID NO:11),HIGKYGLRWRPKNS (SEQ ID NO:56), HIGKYGLRWRPKN (SEQ ID NO:57),HIGKYGLRWRPKGSV (SEQ ID NO:12), HIGKYGLRWRPKGS (SEQ ID NO:58),HIGKYGLRWRPKG (SEQ ID NO:59), HISKYLLRWRPKNSV (SEQ ID NO:60),HISKYLLRWRPKNS (SEQ ID NO:61), HISKYLLRWRPKN (SEQ ID NO:62),HISKYLLRWRPKGSV (SEQ ID NO:63), HISKYLLRWRPKGS (SEQ ID NO:64),HISKYLLRWRPKG (SEQ ID NO:65), HGSKYLLRWRPKNSV (SEQ ID NO:66),HGSKYLLRWRPKNS (SEQ ID NO:67), HGSKYLLRWRPKN (SEQ ID NO:68),HGSKYLLRWRPKGSV (SEQ ID NO:69), HGSKYLLRWRPKGS (SEQ ID NO:70),HGSKYLLRWRPKG (SEQ ID NO:71), HLSKYLLRWRPKNSV (SEQ ID NO:72),HLSKYLLRWRPKNS (SEQ ID NO:73), HLSKYLLRWRPKN (SEQ ID NO:74),HLSKYLLRWRPKGSV (SEQ ID NO:75), HLSKYLLRWRPKGS (SEQ ID NO:76),HLSKYLLRWRPKG (SEQ ID NO:77), HIGKYLLRWRPKNSV (SEQ ID NO:78),HIGKYLLRWRPKNS (SEQ ID NO:79), HIGKYLLRWRPKN (SEQ ID NO:80),HIGKYLLRWRPKGSV (SEQ ID NO:81), HIGKYLLRWRPKGS (SEQ ID NO:82),HIGKYLLRWRPKG (SEQ ID NO:83), HISKYIGRWRPKNSV (SEQ ID NO:84),HISKYIGRWRPKNS (SEQ ID NO:85), HISKYIGRWRPKN (SEQ ID NO:86),HISKYIGRWRPKGSV (SEQ ID NO:87), HISKYIGRWRPKGS (SEQ ID NO:88),HISKYIGRWRPKG (SEQ ID NO:89), HGSKYIGRWRPKNSV (SEQ ID NO:15),HGSKYIGRWRPKNS (SEQ ID NO:90), HGSKYIGRWRPKN (SEQ ID NO:91),HGSKYIGRWRPKGSV (SEQ ID NO:16), HGSKYIGRWRPKGS (SEQ ID NO:92),HGSKYIGRWRPKG (SEQ ID NO:93), HLSKYIGRWRPKNSV (SEQ ID NO:94),HLSKYIGRWRPKNS (SEQ ID NO:95), HLSKYIGRWRPKN (SEQ ID NO:96),HLSKYIGRWRPKGSV (SEQ ID NO:97), HLSKYIGRWRPKGS (SEQ ID NO:98),HLSKYIGRWRPKG (SEQ ID NO:99), HIGKYIGRWRPKNSV (SEQ ID NO:14),HIGKYIGRWRPKNS (SEQ ID NO:100), HIGKYIGRWRPKN (SEQ ID NO:101),HIGKYIGRWRPKGSV (SEQ ID NO:102), HIGKYIGRWRPKGS (SEQ ID NO:103) orHIGKYIGRWRPKG (SEQ ID NO:104) promotes the survival, migration or growthof human adult dermal fibroblasts and/or human adult cardiomyocytes, isresistant to neutrophil elastase and is useful for the treatment ofwounds and/or cardiovascular disease.

In a particular embodiment, each linear or cyclic peptide:HISKYILRWRPKNSV (SEQ ID NO:10) (P46), HIGKYGLRWRPKNSV (SEQ ID NO:11)(NP7), HIGKYGLRWRPKGSV (SEQ ID NO:12) (NP8), HGSKYGLRWRPKNSV (SEQ IDNO:13), HIGKYIGRWRPKNSV (SEQ ID NO:14), HGSKYIGRWRPKNSV (SEQ ID NO:15)or HGSKYIGRWRPKGSV (SEQ ID NO:16) promotes the survival, migration orgrowth of human adult dermal fibroblasts and/or human adultcardiomyocytes, is resistant to neutrophil elastase and is useful forthe treatment of wounds and/or cardiovascular disease.

In another particular embodiment, cyclic HIGKYGLRWRPKGSV (SEQ ID NO:12)(NP8) promotes the survival, migration or growth of human adult dermalfibroblasts and/or human adult cardiomyocytes, is resistant toneutrophil elastase and is useful for the treatment of wounds and/orcardiovascular disease.

All peptides described herein are presented in a linear format ofstandard, single-letter amino acid codes, reading from the N terminus onthe left to the C terminus on the right. “Cyclic” or “cyclized” peptidesmay be represented in linear form but have the N terminus amino acidbound to the C terminus amino acid by one or more standard methods,described below.

A biologically active fibronectin fragment or variant of a FN fragmentdescribed herein is one that functions as a PDGF-BB binding peptide, isresistant to human neutrophil elastase, and functions to a useful extentand in substantially the same manner as the corresponding FN fragment.For example, where a FN fragment having a naturally occurring sequencebinds a GF with a particular affinity and, upon administration to apatient, effectively enhances or alters the GF activity at a site wherethe GF is needed, a biologically active variant of that FN fragment willbe one that, although not identical to the FN fragment, will bind thesame GF(s) with sufficiently useful affinity and similarly enhances oralters the GF(s) at a site of need. For ease of reading, we do notrepeat the term “or a biologically active variant thereof” after everyreference to a FN fragment or other protein or peptide. It is to beunderstood that where FN fragments having a naturally occurring sequenceare useful, so are biologically active variants of those fragments.

With respect to function, a fragment can bind a polypeptide growthfactor with an affinity of at least or about 1×10-7M (e.g., at least1×10-8M; 1×10-9 M; or more). Alternatively, or in addition, a fragmentmay support FN-null cell survival and/or proliferation.

Alternatively, or in addition, the fragment can further include asubstituent at the amino-terminus or carboxy-terminus. The substituentcan be an acyl group or a substituted or un-substituted amine group(e.g., the substituent at the N-terminus can be an acyl group and theC-terminus can be amidated with a substituted or un-substituted aminegroup (e.g., an amino group having one, two, or three substituents,which may be the same or different)). The amine group can be a loweralkyl (e.g., an alkyl having 1-4 carbons). The acyl group can be a loweracyl group (e.g., an acyl group having up to four carbon atoms),especially an acetyl group.

The fragments of fibronectin, including the modified fragments describedabove, can be protease resistant and can include one or more types ofprotecting groups such as an acyl group, an amide group, a benzyl orbenzoyl group, or a polyethylene glycol. More specifically, a fragmentof fibronectin, including the modified fragments described above, can beN-terminally acetylated and/or C-terminally amidated.

The fragments of fibronectin can also be modified in order to improveabsorption, including for example, an addition of sugar residues toenhance transport across the blood-brain barrier.

Any of the fragments can include at least one amino acid residue in theD-form.

Any of the fragments can include at least one non-naturally occurring ormodified amino acid residue (e.g., 4-hydroxyproline,gamma-carboxyglutamic acid, o-phosphoserine, o-phosphotyrosine, ordelta-hydroxylysine). Non-naturally occurring amino acid residues areamino acid residues other than the 20 naturally occurring, geneticallyencoded amino acids. Other examples include naphthylalanine, which canbe substituted for tryptophan to facilitate synthesis, L-hydroxypropyl,L-3,4-dihydroxyphenylalanyl, alpha-amino acids such asL-alpha-hydroxylysyl and D-alpha-methylalanyl, L-alpha-methylalanyl,beta-amino acids, and isoquinolyl. Fragments having non-naturallyoccurring amino acid residues may be referred to as synthetic fragmentsand constitute one type of variant as described herein. Other variantsinclude fragments of fibronectin in which a naturally occurring sidechain of an amino acid residue is replaced with a non-naturallyoccurring side chain (in either the L- or D-form). In another aspect,the invention features polypeptides that include a sequence that isreversed with respect to the N- and C-termini of a sequence naturallyfound in a fibronectin polypeptide or a biologically active variantthereof.

Any of the fragments in the present compositions can be one of aplurality of fragments present. These fragments may be linked togetherby methods described herein. As noted, fragments of fibronectin,including the variant forms described herein, can further include aheterologous polypeptide (i.e., a polypeptide having a sequence thatdoes not appear in a fibronectin). The heterologous polypeptide can be apolypeptide that increases the circulating half-life, cell penetrationor transdermal penetration of the fragment to which it is attached.

The fragments can be contained within physiologically acceptablecompositions, or they may be contained within compositions that are notsuitable for administration to a living being (e.g., concentrated stocksor frozen or lyophilized compositions).

The physiologically acceptable compositions can be pharmaceuticalcompositions, and methods of treating patients are described furtherbelow. The physiologically acceptable compositions can also benon-pharmaceutical compositions or pharmaceutical compositions that canbe dispensed without a physician's prescription. For example, they canbe sold to a distributer or “over the counter” for cosmetic purposes(e.g., to reduce the risk of damage from the skin or to minimize orrepair damage to the skin). For example, the fragments of fibronectinand compositions that include them or combinations of them (e.g., aFN-growth factor complex) can be incorporated in topical formulationssold as cosmetics, moisturizers and the like, sunscreens, shampoos orconditioners, soaps or other foaming cleansers, or lip balm.

The invention also encompasses nucleic acid molecules that encode thepolypeptides described herein. Specific nucleic acid molecules, vectors(e.g., plasmid vectors or viral vectors), and host cells containing themare described further below, as are physiologically acceptablecompositions containing them.

Other compositions of the present invention are tissue engineeredproducts that include a fragment of a fibronectin or a biologicallyactive variant thereof As in other compositions, the fragment or thevariant thereof can bind a polypeptide growth factor or enhance growthfactor activity (as described above and further below), which factor maysubsequently retain biological activity and may be administered to apatient.

Other compositions of the present invention comprise a solid supportthat is associated with (e.g., bound to or impregnated with) one or moreof the fragments of fibronectin, or the biologically active variantsthereof, described herein. The support can be, for example, a tissueculture vessel (e.g., a plate or flask) or device (e.g., a medicaldevice such as one used in wound dressing (e.g., a bandage or gauze),wound repair (e.g., a suture or “steri-strip”), surgical repair (e.g., asurgical mesh), or a tissue implant (e.g. a stent). The fragment offibronectin, or the biologically active variant thereof, can be bound toan active growth factor, including any of those described above.

The methods of the invention include methods for promoting woundhealing. These methods include a step of administering to a patient atherapeutically effective amount of a pharmaceutical compositioncomprising a fragment of fibronectin, or a biologically active variantthereof, as described herein. The fragment of fibronectin, or thebiologically active variant thereof, can be present in a complex withone or more growth factors. The methods can optionally include a step ofidentifying a patient in need of treatment. Such patients includepatients who are suffering from a surgical extirpation or incision ofthe skin, mucosa, underlying connective tissue, fascia, ligament,tendon, cartilage, bone, nerve or muscle; patients who are sufferingfrom a traumatic laceration or tissue loss of the skin, mucosa,underlying connective tissue, fascia, nerve or muscle; and patients whoare suffering from a thermal burn, chemical burn, or ulceration of theskin, mucosa, underlying connective tissue, fascia, nerve or muscle.

As used herein, a “burn” is tissue damage due to exposure to heat or acaustic chemical. A “thermal burn” is tissue damage due to exposure toheat. A “chemical burn” is tissue damage due to exposure to a causticchemical, often strong alkali or strong acid. Agents of chemical burnsto be treated by the peptides defined by the invention include, but arenot limited to, phenol, creosol, mustard gas, phosphorus, nitrogenmustard, arsenic compounds, ammonia, caustic potash, lime, sodiumhydroxide, hydrochloric acid, and sulphuric acid.

The methods of the invention include methods for treating cardiovasculardisease, including decreasing blood vessel occlusion from aggregates ofred blood cell and/or fibrinogen and/or fibrin, as can occur in burnwounds; myocardial infarction; multi-organ failure; diabetes; sicklecell anemia; polycythemia vera; and hyperfibrinogenemia. These methodsinclude a step of administering to a patient a therapeutically effectiveamount of a pharmaceutical composition comprising a fragment offibronectin, or a biologically active variant thereof, as describedherein. The fragment of fibronectin, or the biologically active variantthereof, can be present in a complex with one or more growth factors.The methods can optionally include a step of identifying a patient inneed of treatment.

Suitable formulations are described further below and, generally, takethe form of a solution, lotion, ointment, gel, cream or salve. Thefragments of fibronectin, whether or not complexed with a growth factor,can also be administered by way of their inclusion in an extracellularmatrix (ECM; e.g., a natural or engineered ECM), a bandage, dressing,compress, or the like. By other methods of the invention, one canlocalize an endogenous growth factor to a tissue of a patient. Thesemethods can be carried out by administering, to the patient, atherapeutically effective amount of a composition that includes afragment of fibronectin, or a biologically active variant thereof, asdescribed herein. As in the more specific treatment methods describedabove, these compositions can be administered by way of topicalapplication of a pharmaceutical composition, an engineered ECM, or asolid support. These methods can be described as methods of deliveringone or more growth factors to a patient. The methods can optionallyinclude a step of identifying a patient in need of treatment. Suchpatients include patients who are suffering from an injury to a tissue,a loss of a tissue or a disorder resulting in tissue disfigurement ordysfunction. More specifically, the patient can be suffering from aninjury or loss to the brain, spinal cord or nerves or a disorderresulting in brain, spinal cord or nerve dysfunction; an injury or lossto the heart or blood vessels or a disorder resulting in heart or bloodvessel dysfunction; an injury or loss to the lung, nasopharyngeal tract,sinuses, trachea or airways or a disorder resulting in lung,nasopharyngeal tract, sinus, trachea or airway dysfunction; an injury orloss to the gastrointestinal tract, liver or pancreas or a disorderresulting in gastrointestinal tract, liver or pancreas dysfunction; aninjury or loss to a kidney, ureters, bladder or urethra or a disorderresulting in kidney, ureter, bladder or urethra dysfunction; an injuryor loss to bone, cartilage, synovium, meniscus, ligament, tendon ornucleus pulposus or a disorder resulting in bone, cartilage, synovium,meniscus, ligament, tendon or nucleus pulposus dysfunction; an injury orloss to lips, tongue or gums or a disorder resulting in lip, tongue orgum dysfunction; an injury or loss to the subcutaneous tissue or adisorder resulting in subcutaneous tissue dysfunction.

The invention can also be described in terms of “use,” in which case itencompasses “use” of the compositions described herein, including FNfragments, peptide derivatives of FN fragments, complexes containing oneor more of FN fragments and/or peptide derivatives of FN fragments,including those with a bound GF, nucleic acids encoding the present FNfragments and/or peptide derivatives of FN fragments, expressionvectors, host cells, and tissue engineered products, including thosethat contain biomaterials, for promoting tissue regeneration and/ortissue repair. For example, the present compositions can be used inpromoting wound healing, or for the preparation of a medicament for thepromotion of tissue regeneration or wound healing. The tissueregeneration or repair may result in healing with little or no scarring,in contradistinction with usual adult wound healing.

As used herein, “growth factor binding peptide” (or “GFBP”) and “growthfactor enhancing peptide” (or “GFEP”) are used synonymously.

As used herein, “cosmetic treatment” refers to the use of aphysiologically acceptable composition to improve or maintain theappearance of an individual.

As detailed above, we have found, inter alia, that specific fragments offibronectin and peptides derived from fibronectin can bind variousgrowth factors (e.g., IGF-1, HGF, TGF-β1, TGF-β2, bFGF, FGF-7, PDGF-BB,VEGF-A, or NGF), and the bound growth factors can retain or showenhanced/decreased biological activity. The present invention featurescompositions that include such fragments and peptides, with or withoutbound growth factors in the represented families (i.e., in the IGF, TGF,FGF, PDGF, VEGF, and NGF families), in various formulations andconfigurations. The fragments and peptides may promote synergy with GFsto which the FN fragments or peptides do not bind. In one configuration,the FN fragments or peptides, or FN fragment or peptide/GF-containingcomplexes can be incorporated into engineered two- or three-dimensionalextracellular matrices (which we may abbreviate herein as engECM orrefer to as synthetic matrices), and these can include any of; or anycombination of, the peptides described herein (e.g., a peptideconforming to Formulas I) or biologically active variants thereof Thegrowth factor(s) incorporated can be, for example, IGF-1, TGF-β1,TGF-β2, bFGF, FGF-7, PDGF-BB, VEGF-A, or NGF; any combination orsub-combination thereof; or another specific growth factor in the samefamily as those listed. The growth factors can be exogenously added tothe peptide-containing formulation (e.g., a FN fragment-containingmatrix), or the formulation (e.g., the matrix) can be generated withoutgrowth factors. In the latter case, when placed in the vicinity of anendogenous supply of growth factors, the growth factors can be recruitedby the matrix. The matrix can also recruit cells and induce them todifferentiate, produce tissue or proliferate (presumably by virtue ofthe inclusion or recruitment of growth factors, although the inventionis not limited to compositions that function by any particularmechanism).

The matrix can include any type of biomaterial (e.g., a biopolymer). Forexample, the matrix can be or can include a hydrogel (e.g., anintramolecularly crosslinked hydrogel). The present peptides and GFs canbe incorporated in or associated with many different types of materials(e.g., hyaluronan). The matrix can have, for example, a polycarbonatebackbone, or include biodegradeable polyurethanes. Further examples ofsuitable biopolymers are: proteins (e.g., collagen), protein-containingmacromolecules (e.g., proteoglycans), silk (e.g., a derivatized silk),alginate, chitan and chitosan.

For preparation of pharmaceutical compositions containing one or more ofthe present peptides, for prophylactic and/or therapeutic treatments,the active ingredients (e.g., the peptide alone or the peptide bound toGF(s)) can be incorporated alone or in combination with other activeagents into compositions suitable for administration to a patient. Theformulations can be made using methods routine in the art and particularguidance may be provided by prior formulations of protein-basedtherapeutics. The compositions will be physiologically acceptable (i.e.,substantially non-toxic) and may be formulated as prescriptionmedications or over-the-counter products. Pharmaceuticals orpharmaceutically acceptable compositions contain compounds (e.g.,polypeptides), other materials (e.g., diluents), and/or dosage formsthat are, within the scope of sound medical judgment, suitable for usein contact with the tissues of human beings and animals withoutexcessive toxicity, irritation, allergic response, or other problem orcomplication, commensurate with a reasonable benefit/risk ratio.

Nucleic acid molecules that encode the peptides described herein canalso be formulated for use in cell culture or administration to apatient or subject. Such compositions commonly include apharmaceutically acceptable carrier, and carriers are contemplated inthe present formulations. Any conventional media or agent compatiblewith the active ingredients can be used in the present compositions.While formulations and methods of use are described further below, wenote here that application to human patients is intended, as isapplication to animals (e.g., domesticated, farm, or show animals). Theinvention extends to non-physiologically acceptable compositions in thatit extends to preparatory compositions and compositions suitable forstorage (e.g., concentrated stocks and frozen or lyophilizedpreparations).

The specific sequences described herein are derived from human plasmafibronectin. In addition, one can use corresponding sequences (e.g.,fragments having a corresponding sequence from any fibronectin isoformof any species).

With respect to function, the featured peptides can bind a polypeptidegrowth factor, for example PDGF-BB, with an affinity of about or atleast about 1×10-6-1×10-7 (e.g., about or at least about 5×10-7; 1×10-8;5×10-8; 1×10-9; or 5×10-9). Alternatively or in addition, the peptidessupport FN-null cell survival and/or proliferation secondary tointrinsic growth factor activity and/or growth factor enhancingactivity.

Although applicants do not wish to be bound by theory, the peptidesdescribed herein are useful in the treatment of skin-aging orphoto-aging (e.g., for the treatment of wrinkles) and in other cosmetictreatments in that certain fragments derived from fibronectin have beenshown to promote fibroblast survival and proliferation. Furthermore, thepeptides described herein may be used to deliver growth factors thatpromote fibroblast survival and proliferation to sites needing cosmetictreatment. For example, peptides may be incorporated into transdermalpatches or any other device to facilitate their delivery with or withoutgrowth factors.

Although applicants do not wish to be bound by theory, the peptidesdescribed herein are useful in the treatment of wounds insofar as theystimulate fibroblast survival, proliferation and/or migration.Additionally, the peptides described herein are useful, for example, ascomponents of growth factor delivery devices such as engineeredthree-dimensional extracellular matrices.

Modifications ofPeptides:

The featured fragments and biologically active variants thereof can bemodified in numerous ways. For example, agents, including additionalamino acid residues, other substituents, and protecting groups can beadded to either the amino terminus, the carboxy terminus, or both. Themodification can be made for the purpose of altering the fragments' formor altering the way the fragments bind to or interact with one another,with non-identical fragments, or with other polypeptides. While thepeptides of the present invention may be linear or cyclic, cyclicpeptides generally have an advantage over linear peptides in that theircyclic structure is more rigid and hence their biological activity maybe higher than that of the corresponding linear peptide (see, generally,Camarero and Muir, J. Am. Chem. Soc. 121:5597-5598, 1999).

Strategies for the preparation of circular polypeptides from linearprecursors have been described and can be employed with the presentfragments. For example, a chemical cross-linking approach can be used toprepare a backbone cyclized version of the peptide (Goldenburg andCreighton, J. Mol. Biol., 165:407-413, 1983). Other approaches includechemical intramolecular ligation methods (see, e.g., Camarero et al.,Angew. Chem. Int. Ed., 37:347-349, 1998; Tam and Lu, Prot. Sci.,7:1583-1592, 1998; Camarero and Muir, Chem. Commun., 1997:1369-1370,1997; and Zhang and Tam, J. Am. Chem. Soc. 119:2363-2370, 1997) andenzymatic intramolecular ligation methods (Jackson et al., J. Am. Chem.Soc., 117:819-820, 1995), which allow linear synthetic peptides to beefficiently cyclized under aqueous conditions. See also U.S. Pat. No.7,105,341.

Alternatively, or in addition, any of the present fragments can furtherinclude one or more substituents. For example, the fragment can includea substituent at the amino-terminus, carboxy-terminus, and/or on areactive amino acid residue side-chain. The substituent can be an acylgroup or a substituted or unsubstituted amine group (e.g., thesubstituent at the N-terminus can be an acyl group and the C-terminuscan be amidated with a substituted or unsubstituted amine group (e.g.,an amino group having one, two, or three substituents, which may be thesame or different)). The amine group can be a lower alkyl (e.g., analkyl having 1-4 carbons), alkenyl, alkynyl, or haloalkyl group. Theacyl group can be a lower acyl group (e.g., an acyl group having up tofour carbon atoms), especially an acetyl group. The substituent can be anon-protein polymer, for example, a polyether, a polyethylene glycol(PEG), a polypropylene glycol, or a polyoxyalkylene, a polyalkyleneglycol (for example, polypropylene glycol (PPG), a polybutylene glycol(PBG), or a PPG-PEG block/random polymer. The peptide can be modified bya non-protein polymer by methods known in the art and in the manner setforth in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417;4,791,192 or 4,179,337. The modification (e.g., PEGylation) canstabilize the peptide, reduce its antigenicity, decrease the requireddosage, and/or augment its targeting ability.

The non-protein polymer can vary in size and shape. For example, any ofthe non-protein polymers listed above (e.g., PEG) can be linear,branched, or comb-shaped. Regarding size, the molecular weight can vary.For example, the PEG can have a molecular weight of, for example, about300 kDa, about 1,000 kDa, about 2,000 kDa, about 3,000 kDa, about 4,000kDa, about 5,000 kDa, about 6,000 kDa, about 7,000 kDa, about 8,000 kDa,about 9,000 kDa, about 10,000 kDa, about 11,000 kDa, about 12,000 kDaabout 13,000 kDa about 14,000 kDa about 15,000 kDa, about 20,000 kDa,about 30,000 kDa, about 40,000 kDa, or about 50,000 kDa. For example,the PEG can be of a molecular weight anywhere in between 300 kDA and2000 kDA, 300 kDA and 3000 kDA, 1000 kDA and 2000 kDA and 1000 and 3000kDA.

The non-protein polymer (e.g., PEG) can be linked to the fragment by anynumber of functional group chemistries (e.g., carboxylated-mPEGs,p-nitrophenyl-PEGs, aldehyde-PEGs, amino-PEGs, thiol-PEGs,maleimide-PEGs, aminoxy-PEGs, hydrazine-PEGs, tosyl-PEGs,iodoacetamide-PEGs, succinimidylsuccinate-PEGs,succinimidylglutarate-PEGS, succinimidylcarboxypentyl-PEGs,p-nitrophenycarbonate-PEGs, or ethanethiol-PEGs). The non-proteinpolymer (e.g., PEG) can be linked to the fragment through any number ofchemical groups including, but not limited to, amino-terminal aminoacids, carboxy-terminal amino acids, free amines, and free sulfhydrylgroups.

The non-protein polymer (e.g., PEG) may be a functionalized (forexample, a monofunctional activated linear PEG, a homobifunctionalactivated linear PEG, a heterobifunctional activated linear PEG, amultiarmed activated PEG (e.g., 2-armed, 4-armed, 8-armed, etc.), abranched activated PEG and a comb-shaped activated PEG).

As used herein, the term “alkyl” is meant to refer to a saturatedhydrocarbon group, which is straight-chained or branched. Example alkylgroups include methyl (Me), ethyl (Et), propyl (e.g., n-propyl andisopropyl), butyl (e.g., n-butyl, isobutyl, t-butyl), pentyl (e.g.,n-pentyl, isopentyl, neopentyl), and the like. An alkyl group cancontain from 1 to about 20, from 2 to about 20, from 1 to about 10, from1 to about 8, from 1 to about 6, from 1 to about 4, or from 1 to about 3carbon atoms.

As used herein, “alkenyl” refers to an alkyl group having one or moredouble carbon-carbon bonds. Example alkenyl groups include ethenyl,propenyl, and the like. “Alkynyl” refers to an alkyl group having one ormore triple carbon-carbon bonds. Example alkynyl groups include ethynyl,propynyl, and the like. “Haloalkyl” refers to an alkyl group having oneor more halogen substituents. Example haloalkyl groups include CF3,C2F5, CHF2, CCl3, CHCl2, C2Cl5, and the like.

As used herein, “polyether” refers to a polymer containing etherlinkages. Examples include polyethylene glycol.

The fragments, including the modified fragments described above, can beprotease resistant and can include one or more types of protectinggroups such as an acyl group, an amide group, a benzyl or benzoyl group,or a polyethylene glycol. More specifically, a fragment, including themodified fragments described above, can be N-terminally acetylatedand/or C-terminally amidated.

Where non-naturally occurring or modified amino acid residues areincluded they can be selected from the following or many othersavailable in the art: 4-hydroxyproline, gamma-carboxyglutamic acid,o-phosphoserine, o-phosphotyrosine, or delta-hydroxylysine. Otherexamples include naphthylalanine, which can be substituted fortryptophan to facilitate synthesis, L-hydroxypropyl,L-3,4-dihydroxyphenylalanyl, alpha-amino acids such asL-alpha-hydroxylysyl and D-alpha-methylalanyl, L-alpha-methylalanyl,beta-amino acids, and isoquinolyl. Fragments having non-naturallyoccurring amino acid residues may be referred to as synthetic fragmentsand constitute one type of variant as described herein. Other variantsinclude fragments in which a naturally occurring side chain of an aminoacid residue (in either the L- or D-form) is replaced with anon-naturally occurring side chain.

In one embodiment, the fragments can have three extra amino acids(MetGlySer) at either terminus (or both) (e.g., at the N-terminus) andseven to eight extra amino acids (ThrSerHisHisHisHisHisHisCys) (SEQ IDNO:105) at either terminus (or both) (e.g., at the C-terminus).

For guidance on fragment modification by reduction/alkylation and/oracylation, one can consult Tarr, Methods of ProteinMicrocharacterization, J. E. Silver ed., Humana Press, Clifton N.J.155-194, 1986; for guidance on chemical coupling to an appropriatecarrier, one can consult Mishell and Shiigi, eds, Selected Methods inCellular Immunology, W H Freeman, San Francisco, Calif (1980) and U.S.Pat. No. 4,939,239; and for guidance on mild formalin treatment, one canconsult Marsh, Int. Arch. of Allergy and Appl. Immunol., 41:199-215,1971.

Any of the peptides in the featured compositions can be one of aplurality present in a multimeric form (e.g., a dimer). These multimerscan be linear or branched. The multimeric form can also include one ormore types of fragments and a backbone structure. Where two or morefragments are present, they may be identical or non-identical. A smallerstructure, referred to as a linker, may also be present and may mediateattachment of the fragments to the backbone. Generally, the linker issmaller than the backbone. The nature of the backbone structure is notcritical, and many different types of molecules may be used. One exampleof a linker structure is an oligolysine molecule having, for example,two or more lysine residues (e.g., 2, 3, 4, or more lysine residues).Two or more fragments of the invention (e.g., two three or fourpolypeptides) may be attached to lysine residues by, for example,peptide bonds. These fragments, having a polylysine linker, can belinked to a backbone structure. For example, the invention encompasses:

-   -   Backbone-KKK HIGKYGLRWRPKGSV (SEQ ID NO:106) and    -   HIGKYGLRWRPKGSVKKK-Backbone. (SEQ ID NO:107)

A backbone structure, for example, an oligolysine molecule, may belinear or branched. A multimeric peptide of the invention on a branchedbackbone molecule may be referred to herein as a “dendrimeric” peptide.

Any of the fragments described herein, including the variant formsdescribed herein, can further include a heterologous polypeptide (i.e.,a polypeptide having a sequence that does not appear in a fibronectin).The heterologous polypeptide can be a polypeptide that increases thecirculating half-life of the fragment to which it is attached (e.g.,fused, as in a fusion protein). The heterologous polypeptide can be analbumin (e g , a human serum albumin or a portion thereof) or a portionof an immunoglobulin (e.g., the Fc region of an IgG).

Polypeptide growth factors that can be bound by the FN described hereincan be within the insulin-like growth factor (IGF) family (e.g., IGF-1),within the transforming growth factor (TGF) family (e.g., TGF-β1 orTGF-β2), within the fibroblast growth factor (FGF) family (e.g., bFGF orFGF-7), within the platelet-derived growth factor (PDGF) family (e.g.,PDGF-BB), within the vascular endothelial growth factor (VEGF) subfamily(e.g., VEGF-A), or within the nerve growth factor (NGF) family. Todetermine whether fibronectin fragments bind growth factors that haveretained a biological activity, standard biological assays can becarried out. For example, as outlined in the Examples below, migratoryresponses to bound growth factors that usually stimulate migration canbe carried out. For example, one can compare the effect of a bound andunbound growth factor on fibroblast migration or granulation tissueformation. Specifically, if a growth factor is a PDGF (e.g., PDGF-BB),migration of AHDF cells can be analyzed.

Compounds mimicking the necessary conformation of the fibronectinfragments described herein are contemplated as within the scope of thisinvention. A variety of designs for such mimetics are possible. U.S.Pat. Nos. 5,192,746; 5,169,862; 5,539,085; 5,576,423; 5,051,448; and5,559,103, all hereby incorporated by reference, describe multiplemethods for creating such compounds.

Physiologically Acceptable Compositions:

A present pharmaceutical composition is formulated to be compatible withits intended route of administration, for example, oral or parenteral(e.g., intravenous, intradermal, subcutaneous, intraperitoneal,intramuscular, by inhalation, transdermal (topical), and transmucosaladministration). Given the ability of the present FN fragments, andGF-containing complexes bearing these fragments, to facilitate woundhealing, topical formulations are particularly envisioned.

Solutions or suspensions used for parenteral administration can include:a sterile diluent such as water for injection, saline solution, fixedoils, polyethylene glycols, glycerine, propylene glycol or othersynthetic solvents; antibacterial agents such as benzyl alcohol ormethyl parabens; antioxidants such as ascorbic acid or sodium bisulfate;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose. pH can be adjusted withacids or bases, such as hydrochloric acid or sodium hydroxide. Thecomposition can be aliquoted or packaged in ampules, disposablesyringes, single or multiple dose vials made of glass or plastic,bottles, and the like, and such packaged forms, along with instructionsfor use, are within the scope of the present invention. Preferably, thecompositions are sterile at a medically acceptable level in view of theintended route of administration.

Pharmaceutical compositions adapted for injection include, for example,sterile aqueous solutions (where water soluble) or dispersions andsterile powders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include, for example, physiological saline, bacteriostaticwater, Cremophor EL™ (BASF, Parsippany, N.J.) and phosphate bufferedsaline (PBS). In all cases, the compositions prepared for administrationshould be sterile and should be fluid or convertible to a fluid at leastsufficient for easy syringability. The composition and/or nucleic acidconstructs should be stable under the conditions of manufacture andstorage and should be preserved against the contaminating action ofmicroorganisms such as bacteria and fungi. Preservatives againstmicroorganisms can include various antibacterial and antifungal agents,for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal,and the like.

The carrier can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), and suitablemixtures thereof. Fluidity can be maintained, for example, by the use ofa coating such as lecithin, by the maintenance of the required particlesize in the case of dispersions and by the use of surfactants.

In many cases, it will be desirable for the composition to be isotonicto blood. This can be accomplished using various isotonic agents, forexample, sugars, polyalcohols such as manitol, sorbitol, sodium chloridein the composition.

Delayed or extended absorption of the injectable compositions can bedesirable and can be achieved by including in the composition an agentwhich delays absorption, for example, aluminum monostearate and gelatin,or by coating micro- or nano-particles of active agent in thecomposition with materials that delayed or extended release ofcomponents.

Sterile injectable solutions can be prepared, for example, bysolubilizing or suspending the active compound in the required amount inan appropriate solvent with one or a combination of additionalingredients. Typically creation of such solution or suspension isfollowed by sterile filtration. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the other desired ingredients. In the caseof sterile powders for the preparation of sterile injectable solutions,the preparation is dried, e.g., by vacuum drying and/or freeze-drying.

Pharmaceutical compositions adapted for topical administration mayinclude, but are not limited to, compositions in the form of skin care,skin cleansing, or anti-wrinkle products, shampoos, make-up,conditioners, lotions, aerosols, gels, mousses, dyes, or bleaches. Thesecompositions may contain one or more conventional cosmetic ordermatological additives or adjuvants, including, but not limited to,fillers, surfactants, thixotropic agents, antioxidants, preservingagents, dyes, pigments, fragrances, thickeners, vitamins, hormones,moisturizers, UV absorbing organic sunscreens, UV scattering inorganicsunscreens, wetting agents, cationic, anionic, nonionic or amphotericpolymers, and hair coloring active substances. These adjuvants are wellknown in the field of cosmetics and are described in many publications,for example see Harry's Book of Cosmeticology, 8^(th) edition, MartinRieger, ed., Chemical Publishing, New York (2000). Exemplarycompositions are described in, for example, in U.S. Pat. Application2005008604, U.S. Pat. Application 20050025725 and U.S. Pat. Application20040120918 which are herein incorporated by reference.

In certain embodiments, the pharmaceutical compositions of thisinvention can include one or more chemical penetration enhancers (asdescribed, for example, in International Publication No. WO2005009510).

Exemplary chemical penetration enhancers include, but are not limitedto, 1-dodecyl pyrrolidone, benzyl dimethyl dodecyl ammonium chloride,cocamidopropyl betaine, cocamidopropyl hydroxysultaine, oleyl betaine,cineole, cetyl trimethyl ammonium bromide, dodecyl amine, dodecylpyridinium chloride, hexadecyl trimethyl ammoniopropane sulfonate,isopropyl myristate, lauric acid, limonene, linoleic acid, linolenicacid, menthol (terpene), methyl laurate, 1-methyl-2-pyrrolidone,N-lauryl sarcosine (CAS number 137-16-6, also called sodium lauroylsarcosinate), nicotine sulfate, oleic acid, octyl trimethyl ammoniumbromide, polyethyleneglycol dodecyl ether, 1-phenyl piperazine, sorbitanmonolaurate, sodium lauryl ether sulfate, sodium dodecyl sulfate, sodiumoleate, sodium octyl sulfate, tetracaine, and Tween-20™.

Chemical penetration enhancers increase skin permeability and are knownin the art (see, for example, Shah et al. “Skin Penetration Enhancement:Clinical pharmacological and regulatory considerations.” PharmaceuticalSkin Penetration Enhancement, ed. K. Walters. 1993, New York, Basel,Hong Kong: Marcel Dekker. 417-427).

The present peptides may be used in cosmetic compositions either as thepeptides themselves or in the form of a premix in a suitable excipientand they may be used in the form of a solution, dispersion, emulsion,paste or powder. They may individually or with other active substances,including but not limited to those specifically described herein, becarried by cosmetic vectors such as macro-, micro- or nanocapsules,liposomes or chylomicrons, macro-, micro- or nanoparticles ormicrosponges. They may also be adsorbed on powdered organic polymers,talcs, bentonites and other inorganic carriers.

The peptides may be used in any form or in a form that is bound,incorporated, absorbed in or adsorbed on macro-, micro- andnanoparticles, macro-, micro- and nanocapsules for the treatment oftextiles, synthetic or natural fibers, wools and all materials liable tobe used in the manufacture of clothing or underwear for the day ornight, intended for contact with the skin, such as pantyhose, underwear,handkerchiefs and wipes, in order to exert a cosmetic effect through thecontact between the textile and skin and enable continuous topicaldelivery.

The peptides can be used in topical compositions (e.g., therapeutic orcosmetic compositions) at concentrations ranging from 0.00001% (w/w)(“w/w” is weight/weight) and 10% (w/w) (e.g., between about 0.0001%(w/w) and 1% (w/w)). Another useful range is from about 0.001% and about5% (w/w). The peptides may also be used in the range of about 1 ppm toabout 500 ppm (e.g., about 100 to about 400 ppm).

Compositions for oral administration typically include an inert oredible diluent or edible carrier. Such compositions can be formulated invarious ways, e.g., in liquid, capsule, or tablet form. Pharmaceuticallycompatible binding agents, and/or adjuvant materials can be included aspart of the composition. The tablets, pills, capsules, troches and thelike can contain any one or more of the following ingredients, orcompounds of a similar nature: a binder such as microcrystallinecellulose, gum tragacanth or gelatin; an excipient such as starch orlactose, a disintegrating agent such as alginic acid, Primogel, or cornstarch; a lubricant such as magnesium stearate or Sterotes; a glidantsuch as colloidal silicon dioxide; a sweetening agent such as sucrose orsaccharin; or a flavoring agent such as peppermint, methyl salicylate,or orange flavoring.

For inhalation administration (e.g., for application to wounded tissues,such as mucosa, within the nasal passages, nasopharynx, trachea or lungsor), the present compositions are delivered in the form of a wet or dryaerosol spray, e.g., from a pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalroutes. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are typically used in theformulation. A number of such penetrants are generally known in the art,and include, for example, for transmucosal administration, detergents,bile salts, and fusidic acid derivatives. Administration may also befacilitated by iontophoresis, microneedles and other devices designed toenhance transdermal penetration.

Transmucosal administration can be accomplished through the use of nasalsprays or suppositories (e.g., using conventional suppository bases suchas cocoa butter and other glycerides). For transdermal administration,the active compounds are formulated into ointments, salves, gels, orcreams as generally known in the art.

Such compositions can also be formulated with carriers that will protectthe compositions against rapid elimination from the body, such as acontrolled release formulation, including implants and microencapsulateddelivery systems. Biodegradable, biocompatible polymers can be used,such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid,collagen, polyorthoesters, polycarbonates, and polylactic acid. Thematerials can also be obtained commercially, e.g., from Alza Corporationand Nova Pharmaceuticals, Inc. Liposomal suspensions (includingliposomes targeted to particular cells (e.g., targeted to infectedcells) with monoclonal antibodies) can also be used to preparepharmaceutical compositions. These can be prepared according to methodsknown to those skilled in the art, for example, as described in U.S.Pat. No. 4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals. In one embodiment of the fibronectin fragmentsand peptide derivatives of fibronectin fragments of the invention, thedosage unit form is about 0.1 to 5 mg of lyophilized peptide or peptidederivative. In another embodiment of the fibronectin fragments andpeptide derivatives of fibronectin fragments of the invention, thedosage unit form is about 1 mg of lyophilized peptide or peptidederivative.

Toxicity and therapeutic efficacy of active compounds and pharmaceuticalcompositions can be determined by standard pharmaceutical procedures incell cultures or experimental animals. For example, such procedures areroutinely applied for determining the LD50 (the dose lethal to 50% ofthe population) and the ED50 (the dose therapeutically effective in 50%of the population). The dose ratio between toxic and therapeutic effectsis the therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds that exhibit large therapeutic indices are generallypreferred. The data obtained from the cell culture assays and animalstudies (including those described in the examples, below) can be usedin formulating a range of dosage for use in humans or other intendedsubjects. The dosage of such compounds is usually selected to produce arange of circulating concentrations that include the ED50 with little orno toxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. For anycompound used in the method of the invention, the therapeuticallyeffective dose can be estimated initially from cell culture assays.Thus, for example, a dose may be initially established in animal modelsto achieve a circulating plasma concentration range that includes theEC50 (i.e., the concentration of the test compound which achieves ahalf-maximal response) as determined in cell culture. Such informationcan be used to more accurately determine useful doses in humans. Levelsin plasma may be measured, for example, by high performance liquidchromatography, or by other suitable analysis method adapted for thecompound of interest.

As noted, peptides (e.g., synthetic or recombinantly produced peptides)with growth factor-binding and/or—enhancing/inhibiting activity can beincorporated into a tissue engineered product. FN domains that promotefibroblast migration can also be included. Preferably, the products arerobust (i.e., relatively resistant to rapid degradation). They can beused, for example, in treating wounds, including acute or non-healingwounds (e.g., chronic ulcers). Patients amenable to treatment aredescribed further below. Alternatively or in addition, growthfactor-binding and/or enhancing peptides can be tethered to abiocompatible polymer for delivery of one or more growth factors to acell, tissue or organ in need of treatment or for endogenouslocalization of growth factors. Alternatively or in addition, growthfactor-binding and/or enhancing peptides can be incorporated in apolymer or nonpolymer biomaterial for controlled release to an acute ornon-healing wound.

We have developed an engineered ECM that is conductive and inductive ofnew tissue formation in porcine cutaneous wounds utilizing moleculardomains C, H, and HV or cell adhesion peptides of the blood proteinfibronectin (FN) tethered to an intramolecularly crosslinked hyaluronan(HA) hydrogel. Thus, in one implementation, the invention includes anengineered ECM that includes a fragment of a fibronectin (e.g., a plasmafibronectin) or a biologically active variant thereof The fragment canbe tethered to (e.g., covalently or non-covalently bound to) a hydrogel(e.g., an HA hydrogel) and can be a fragment that binds and/or enhancesa polypeptide growth factor. The fragment can be tethered according toattachment methods discussed in U.S. Pat. Application 20050282747, thecontents of which are incorporated herein in their entirety.

The naturally-occurring ECM is comprised of diverse constituents such asglycoproteins, proteoglycans, complex carbohydrates, and othermolecules. Major functions of the ECM include, but are not limited to,providing structural support, tensile strength or cushioning; providingsubstrates and pathways for cell adhesion and cell migration; andregulating cellular differentiation and metabolic function. ECM proteinsinclude, for example, collagens, elastin, fibronectin, laminin,proteoglycans, vitronectin, thrombospondin, tenascin (cytoactin),entactin (nidogen), osteonectin (SPARC), anchorin CII, chondronectin,link protein, osteocalcin, bone sialoprotein, osteopontin, epinectin,hyaluronectin, amyloid P component, fibrillin, merosin, s-laminin,undulin, epilligrin, and kalinin.

The featured tissue engineered product (e.g., the engineered ECM) caninclude biological and/or synthetic components. It can include abiopolymer (e.g., hyaluronan (HA), a glycosaminoglycan (GAG),fibrinogen, laminin, or collagen). The biocompatible polymer can be asynthetic biodegradable polymer, many of which are known in the art. Forexample, the biodegradable polymer can be a poly(lactide), apoly(glycolide), a poly(lactide-coglycolide), a poly(lactic acid), apoly(glycolic acid), a poly(lactic acid-co-glycolic acid), apoly(caprolactone), a polycarbonate, a polyesteramide, a polyanhydride,a poly(amino acid), a poly(ortho ester), a polycyanoacrylate, apolyamide, a polyacetal, a poly(ether ester), a copolymer ofpoly(ethylene glycol) and a poly(ortho ester), a poly(dioxanone), apoly(alkylene alkylate)s, a biodegradable polyurethane, or any blend orcopolymer thereof. Other useful polymers include an alginate polymer anda carboxy-vinyl polymer (e.g., a polymer including at least 90% acrylicacid monomers and about 0.1% to about 5.0% of a difunctionalcrosslinking agent).

A tissue engineered “smart” matrix that would be conductive andinductive of tissue cell repopulation of a wound site and thedevelopment of new tissue, respectively, can be composed of GFs, oractive fragments thereof, in the context of an appropriate ECM that arerequired for optimal wound repair. In addition, FN GF-binding domain(s)may provide a useful tool for engineering many other GF localization(from endogenous or exogenous sources) and/or delivery systems for softor hard tissue repair, augmentation and regeneration. Furthermore,growth factor FN/VN-binding peptides or molecularly engineeredderivatives of the FN and VN GF-binding domains might become stronglyinhibitory of GF activity and thus useful for proliferative or fibroticdisorders such as cancer, pulmonary fibrosis, GI or GU stenosis, burncontractures and autoimmune generated sclerosis.

EngECM can be generated with or without growth factors, or activefragments thereof (e.g., growth factors and fragments described herein).In the former case, the dosage of growth factors in the engECM can vary,e.g., as described below, 100 ng/ml (15 ng total per wound) of PDGF-BBadded to 2:1 engineered ECM enhanced granulation formation at 4 daysafter injury and application of material. In the latter case, whenplaced in the vicinity of an endogenous supply of growth factors, thegrowth factors can be recruited by the matrix.

The invention further encompasses nucleic acid molecules, including DNAand RNA molecules, that encode the polypeptides described herein. Thenucleic acid molecules can be formulated in physiologically acceptablecompositions for administration. The invention also features vectorsthat include the present nucleic acid constructs. Of particular benefitare expression vectors, especially those for expression in eukaryoticcells. Such vectors can, for example, be viral, plasmid, cosmid, orartificial chromosome (e.g., yeast artificial chromosome) vectors.Typically, plasmids are circular, dsDNA elements that include one ormore cloning sites for insertion of selected DNA sequences, e.g., codingsequences. Such plasmids may include a functional origin of replicationand thus are replication competent, or may be replication defective.

In addition to plasmids, viral vectors (e.g., replication defectiveretroviruses, lentiviruses, adenoviruses and adeno-associated viruses)can also be advantageously used. A large number of such viral vectorshave been developed having a broad variety of different properties. Forexample, such viral vectors may be replication defective retroviruses,adenoviruses and adeno-associated viruses. Techniques and procedures forproducing recombinant retroviruses and for infecting cells in vitro orin vivo with such viruses are provided in Current Protocols in MolecularBiology, Ausubel, F. M. et al. (eds.) Greene Publishing Associates,(1989), Sections 9.10-9.14 and other standard laboratory manuals.Examples of suitable retroviruses include pLJ, pZIP, pWE and pEM whichare well known to those skilled in the art. Examples of suitablepackaging virus lines include psi.Crip, psi.Cre, psi.2 and psi.Am. Thegenome of adenovirus can be manipulated such that it encodes andexpresses a nucleic acid construct, as described herein, but isinactivated in terms of its ability to replicate in a normal lytic virallife cycle. (see, e.g., Berkner et al., BioTechniques 6:616, 1988;Rosenfeld et al., Science 252:431-434, 1991; and Rosenfeld et al., Cell68:143-155, 1992). Suitable adenoviral vectors derived from theadenovirus strain Ad type 5 d1324 or other strains of adenovirus (e.g.,Ad2, Ad3, Ad7 etc.) are well known to those skilled in the art.Alternatively, an adeno-associated virus vector such as that describedin Tratschin et al. (Mol. Cell. Biol. 5:3251-3260, 1985) can be used toexpress a transactivator fusion protein. Other viral vector alternativesinclude lentiviral vectors. Such vectors and their preparation and useare described, for example, in U.S. Pat. Nos. 6,924,123; 6,863,884;6,830,892; 6,818,209; 6,808,923; 6,799,657, all of which areincorporated herein in their entireties.

The vectors of the invention can advantageously include a polypeptidefragment described herein. Other elements included in the design of aparticular expression vector can depend on such factors as the choice ofthe host cell to be transformed, the level of expression of proteindesired, etc. The expression vectors of the invention can be introducedinto host cells to thereby produce proteins or peptides, includingfusion proteins or peptides, encoded by nucleic acids as describedherein.

The vectors described herein can be introduced into cells or tissues byany one of a variety of known methods within the art. Such methods aredescribed for example in Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, New York (1992), whichis hereby incorporated by reference. See, also, Ausubel et al., CurrentProtocols in Molecular Biology, John Wiley and Sons, Baltimore, Md.(1989); Hitt et al., “Construction and Propagation of Human AdenovirusVectors,” in Cell Biology: A Laboratory Handbook, Ed. J. E. Celis.,Academic Press. 2nd Edition, Volume 1, pp: 500-512, 1998; and Hitt etal., “Techniques for Human Adenovirus Vector Construction andCharacterization,” in Methods in Molecular Genetics, Ed. K. W. Adolph,Academic Press, Orlando, Fla., Volume 7B, pp: 12-30, 1995. The methodsinclude, for example, stable or transient transfection, lipofection,electroporation and infection with recombinant viral vectors. The term“transfecting” or “transfection” is intended to encompass allconventional techniques for introducing nucleic acid into host cells,including calcium phosphate co-precipitation, DEAF-dextran-mediatedtransfection, lipofection, electroporation and microinjection. Suitablemethods for transfecting host cells can be found in Sambrook et al.(Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring HarborLaboratory press (1989)), and other laboratory textbooks.

For plant cells, a Ti plasmid or viral vector is often used. Forexample, such plasmids and viral vectors can be used to transfect hostplant cells via Agrobacterium tumefaciens-mediated transfection (forplant cells susceptible to A. tumefaciens infection), or can be directlyinserted in cells, e.g., using microinjection, particle bombardment, orelectroporation. In other methods, protoplasts can be made from plantcells and then transfected.

The number of host cells transformed with a nucleic acid constructs ofthe invention will depend, at least in part, upon the type ofrecombinant expression vector and the type of transfection techniqueused. Nucleic acid can be introduced into a host cell transiently, orfor long-term expression. For long-term expression, the nucleic acid isstably integrated into the genome of the host cell or remains as astable episomal element.

For integration of nucleic acid into host cell DNA, typically a gene isused that encodes a selectable marker (e.g., drug resistance) isintroduced into the host cells along with the nucleic acid of interest.A variety of such selectable markers are commonly used, such as thedrugs hygromycin and neomycin. Selectable markers can be introduced on aseparate plasmid or other vector from the nucleic acid of interest or,are introduced on the same vector. Host cells transfected with a nucleicacid construct of the invention (e.g., a recombinant expression vector)and a gene for a selectable marker can be identified by selecting forcells using the selectable marker.

The present nucleic acid constructs can be introduced into eukaryoticcells growing in culture in vitro by conventional transfectiontechniques (e.g., calcium phosphate precipitation, DEAE-dextrantransfection, electroporation, and other methods). Cells can also betransfected in vivo, for example by application of a delivery mechanismsuitable for introduction of nucleic acid into cells in vivo, such asviral vectors (see e.g., Ferry et al., Proc. Natl. Acad. Sci. USA88:8377-8381, 1991, and Kay et al., Human Gene Therapy 3:641-647, 1992),adenoviral vectors (see e.g., Rosenfeld, Cell 68:143-155, 1992; and Herzand Gerard, Proc. Natl. Acad. Sci. USA 90:2812-2816, 1993),receptor-mediated DNA uptake (see e.g., Wu and Wu, J. Biol. Chem.263:14621, 1988; Wilson et al., J. Biol. Chem. 267:963-967, 1992; andU.S. Pat. No. 5,166,320), direct injection of DNA (see e.g., Acsadi etal., Nature 332:815-818, 1991; and Wolff et al., Science 247:1465-1468,1990) or particle bombardment (see e.g., Cheng et al., Proc. Natl. Acad.Sci. USA 90:4455-4459, 1993; and Zelenin et al., FEBS Letters 315:29-32,1993). Thus, in the present invention, cells can be transfected in vitroor ex vivo, and the expressed peptide can be isolated there from bymethods known in the art. The cells can also be administered to asubject or, alternatively, cells can be directly modified in vivo. Inany of these situations, the nucleic acid construct used to express thepeptide can include a signal sequence to facilitate export from thecell.

Another aspect of the invention pertains to host cells into which anucleic acid construct of the invention has been introduced, i.e., a“recombinant host cell.” It is understood that the term “recombinanthost cell” refers not only to the particular subject cell but to theprogeny or potential progeny of such a cell. Because certainmodifications may occur in succeeding generations due to either mutationor environmental influences, such progeny may not, in fact, be identicalto the parent cell, but are still included within the scope of the termas used herein.

A host cell can be any prokaryotic or eukaryotic cell, althougheukaryotic cells are preferred. Exemplary eukaryotic cells includemammalian cells (such as Chinese hamster ovary cells (CHO) or COScells). Other suitable host cells are known in the art.

It is not intended that the present invention be limited by theparticular nature of the therapeutic preparation, so long as thepreparation comprises an appropriate fragment of fibronectin that bindsa polypeptide growth factor or that has intrinsic survival or growthfactor activity or an appropriate fragment of a growth factor that bindsfibronectin. For example, such compositions can be provided togetherwith physiologically tolerable liquid, gel or solid carriers, diluents,adjuvants and/or excipients.

These therapeutic preparations can be administered to mammals forveterinary use, such as with domestic animals, and clinical use inhumans in a manner similar to other therapeutic agents. In general, thedosage required for therapeutic efficacy will vary according to the typeof use and mode of administration, as well as the particularizedrequirements of individual hosts.

Such compositions are typically prepared as liquid solutions orsuspensions, or in solid forms. Formulations can include such normallyemployed additives such as binders, fillers, carriers, preservatives,stabilizing agents, emulsifiers, buffers and excipients as, for example,pharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharin, cellulose, magnesium carbonate, and the like. Thesecompositions take the form of solutions, suspensions, tablets, pills,capsules, sustained release formulations, or powders, and typicallycontain 1%-95% of active ingredient, preferably 2%-70%.

The compositions are also prepared as injectables, either as liquidsolutions or suspensions; solid forms suitable for solution in, orsuspension in, liquid prior to injection may also be prepared.

The fragments of the present invention are often mixed with diluents orexcipients which are physiologically tolerable and compatible. Suitablediluents and excipients are, for example, water, saline, dextrose,glycerol, or the like, and combinations thereof. In addition, if desiredthe compositions may contain minor amounts of auxiliary substances suchas wetting or emulsifying agents, stabilizing or pH buffering agents.

Additional formulations which are suitable for other modes ofadministration, such as topical administration, include salves,tinctures, creams, lotions, and, in some cases, suppositories. Forsalves and creams, traditional binders, carriers and excipients mayinclude, for example, polyalkylene glycols or triglycerides.

Methods of Use:

The fibronectin fragments and peptide derivatives of fibronectinfragments described herein are useful in promoting tissue regeneration,e.g., wound healing, and in cosmetic and therapeutic formulations forthe prevention and treatment of poor skin appearance related to, forexample, aging. Use in cell culture is also described. The polypeptides(or nucleic acids or expression vectors encoding them or cellsexpressing them) can be incorporated into, for example, therapeuticformulations for the indications described herein as well as intoproducts and compositions for improving, for example, skin appearanceand/or feel of skin exhibiting signs of skin aging.

For example, compositions of the present invention are useful forregulating the appearance of skin due to wrinkles and UVB photodamage byproviding visual improvement in skin appearance following application ofthe composition to the skin. Generally speaking, compositions of thepresent invention which further contain particulate materials will bemost useful for providing the immediate visual improvement.

The invention features cosmetic treatments including those forprophylactically regulating a skin condition and those fortherapeutically regulating a skin condition. “Signs of skin aging,”“poor skin appearance,” and other phrases similarly referring to, forexample, symptoms of aging and the like include, but are not limited to,all outward visibly and tactilely perceptible manifestations as well asany other macro or micro effects due to skin aging. Such signs may beinduced or caused by intrinsic factors and/or extrinsic factors, e.g.,chronological aging and/or environmental damage (e.g., UVB photodamage,exposure to pollutants, and poor diet). These signs may result fromprocesses which include, but are not limited to, the development oftextural discontinuities such as wrinkles and coarse deep wrinkles, skinlines, crevices, bumps, large pores (e.g., associated with adnexalstructures such as sweat gland ducts, sebaceous glands, or hairfollicles), or unevenness or roughness, loss of skin elasticity (lossand/or inactivation of functional skin elastin), sagging (includingpuffiness in the eye area and jowls), loss of skin firmness, loss ofskin tightness, loss of skin recoil from deformation, discoloration(including undereye circles), blotching, sallowness, hyperpigmented skinregions such as age spots and freckles, keratoses, abnormaldifferentiation, hyperkeratinization, elastosis, collagen breakdown, andother histological changes in the stratum corneum, dermis, epidermis,the skin vascular system (e.g., telangiectasia or spider vessels), andunderlying tissues, especially those proximate to the skin. Particularlypreferred in accordance with the present invention, the signs of skinaging are wrinkles and the compositions of the present invention are, incertain preferred embodiments, useful in fighting, treating orpreventing wrinkles.

Wrinkles can result from numerous causes. For example, wrinkles can becaused from the natural aging process of the skin, from smoking, andfrom exposure to ultraviolet radiation (e.g., from chronic sunexposure). Wrinkles can be classified as described in Kligman et al.(Br. J. Derm. 113:37-42, 1985), herein incorporated by reference.Kligman classifies wrinkles into three classes: linear wrinkles, glyphicwrinkles, and crinkles, and any of these types of wrinkles, regardlessof their cause, can be treated as described herein. Aside from wrinklesper se, the present compositions can be used to improve the skin'sappearance.

The methods disclosed herein are useful to prevent or treat or reducewrinkles, including UV-induced wrinkles, and/or to improve skin qualityand appearance in a subject. The methods can be carried out byadministering to the subject a composition containing a fibronectinfragment or a biologically active variant thereof An exemplary treatmentmethod can include locating a wrinkle or a potential site of wrinklingand applying a composition described herein.

As used herein, prophylactically regulating a skin condition includesdelaying, minimizing and/or preventing visible and/or tactilediscontinuities in skin (e.g., texture irregularities in the skin whichmay be detected visually or by feel), including signs of skin aging.

As used herein, therapeutically regulating skin condition includesameliorating, e g , diminishing, minimizing and/or effacing,discontinuities in skin, including signs of skin aging. Some of theproducts produced using the compositions of the present invention andindeed the compositions themselves may be used for prophylactically ortherapeutically regulating a skin condition.

In certain preferred aspects, the present invention is useful forimproving the physiological state and/or the physical appearance ofhuman skin, in particular to reduce the signs of skin aging that aregenerated by sun exposure (e.g., UVB photodamage), physical and hormonalstress, abrasion, nutritional effects and other similar causes. Thecompositions may often be used to prevent the signs of aging and/or totreat them in order to afford the consumer who uses them, a moreyouthful appearance.

All terms such as “skin aging,” “signs of skin aging,” “poor skinappearance,” “topical application,” and the like are used in the sensein which they are generally and widely used in the art of developing,testing and marketing cosmetic and personal care products. “Wrinkles”means furrows in the otherwise smooth surface of the facial skin,visible to the naked eye, in the average depth of 50 to more than 200 μmand essentially appearing with progressive age. The term “cosmeticcomposition” in accordance with the present invention relates to aformulation that can be used for cosmetic purposes, purposes of hygieneor as a basis for delivery of one or more pharmaceutical ingredients.This includes cosmetics, personal care products and pharmaceuticalpreparations. It is also possible that these formulations are used fortwo or more of these same purposes at one time. A medicated dandruffshampoo, for example, has pharmacological properties and is used as apersonal care product to provide clean hair. These compositions may alsoinclude additional ingredients such as a dermatologically acceptablecarrier. “Cosmetics,” as used herein, include without limitation,lipstick, mascara, rouge, foundation, blush, eyeliner, lipliner, lipgloss, facial or body powder, sunscreens and blocks, nail polish,mousse, sprays, styling gels, nail conditioner, whether in the form ofcreams, lotions, gels, ointments, emulsions, colloids, solutions,suspensions, compacts, solids, pencils, spray-on formulations, brush-onformulations and the like. “Personal care products” include, withoutlimitation, bath and shower gels, shampoos, conditioners, cream rinses,hair dyes and coloring products, leave-on conditioners, sunscreens andsunblocks, lip balms, skin conditioners, cold creams, moisturizers, hairsprays, soaps, body scrubs, exfoliants, astringents, depilatories andpermanent waving solutions, antidandruff formulations, antisweat andantiperspirant compositions, shaving, preshaving and after shavingproducts, moisturizers, deodorants, cold creams, cleansers, skin gels,rinses, whether in solid, powder, liquid, cream, gel, ointment, lotion,emulsions, colloids, solutions, suspensions, or other form.“Pharmaceutical preparations” in accordance with the present inventioninclude, without limitation, carriers for dermatological purposes,including topical and transdermal application of pharmaceutically activeingredients. These can be in the form of gels, patches, creams, nosesprays, ointments, lotions, emulsions, colloids, solutions, suspensions,powders and the like. Compositions in accordance with the inventioninclude cosmetics, personal care products and pharmaceuticalpreparations.

The invention features methods for promoting tissue regeneration,including, for example, wound healing. As used herein, tissueregeneration is used to refer to the replacement of damaged tissue bythe proliferation and differentiation of cells into a tissue. Tissuedamage can occur by any means, including physical injury, disease, andinfection. As described herein, “wound-healing” is used as anon-limiting example of tissue regeneration.

The primary goal in the treatment of wounds is to achieve wound closure.Open cutaneous wounds represent one major category of wounds and includethermal and/or chemical burn wounds, neuropathic ulcers, pressure sores,venous stasis ulcers, and diabetic ulcers. Open cutaneous woundsroutinely heal by a process which comprises six major components: i)inflammation, ii) fibroblast proliferation, iii) blood vesselproliferation, iv) connective tissue synthesis v) epithelialization, andvi) wound contraction. Wound healing is impaired when these components,either individually or as a whole, do not function properly. Numerousfactors can affect wound healing, including malnutrition, infection,pharmacological agents (e.g., actinomycin and steroids), diabetes, andadvanced age (see Hunt and Goodson in Current Surgical Diagnosis &Treatment (Way; Appleton & Lange), pp. 86-98, 1988).

The term “wound” refers broadly to injuries to the skin and subcutaneoustissue initiated in different ways (e.g., pressure sores from extendedbed rest and wounds induced by trauma) and with varying characteristicsas well as to injuries of other tissues and bone, including tissues andbone in or around the vicinity of a primary wound site. Of course,wounds can also be made surgically or by disease (e.g. cancer). Woundsmay be classified into one of four grades depending on the depth of thewound: i) Grade I: wounds limited to the epithelium; ii) Grade II:wounds extending into the dermis; iii) Grade III: wounds extending intothe subcutaneous tissue; and iv) Grade IV (or full-thickness wounds):wounds wherein bones are exposed (e.g., a bony pressure point such asthe greater trochanter or the sacrum). The term “partial thicknesswound” refers to wounds that encompass Grades I-III; examples of partialthickness wounds include thermal or chemical burn wounds, pressuresores, venous stasis ulcers, and diabetic ulcers. The term “deep wound”is meant to include both Grade III and Grade IV wounds. The presentinvention contemplates treating all wound types, including deep woundsand chronic wounds.

The phrases “promote wound healing,” “enhance/improve wound healing,”and the like refer to either the induction of the formation ofgranulation tissue and/or the induction of epithelialization (i.e., thegeneration of new cells in the epithelium), and/or reduction ofscarring. Wound healing is conveniently measured by decreasing woundarea. It is not intended that phrases such as “promote wound healing” or“enhance/improve wound healing” require a quantitative comparison withcontrols. In the case of treatment of a chronic wound, it is sufficientthat evidence of wound healing begins after treatment. Many traumaticwounds and cancer extirpations must be left open to heal by secondaryintention, and patients having such wounds and extirpations can betreated with the compositions described herein that promote woundhealing.

The phrase “therapeutically effective amount” of the fibronectinfragments or peptide derivatives of fibronectin fragments of theinvention, when referring to wound healing, promoting wound healing orenhancing wound healing, is that amount that promotes induction of theformation of granulation tissue and/or the induction ofepithelialization and/or reduction of scarring. For example, fibronectinfragments or peptide derivatives of fibronectin fragments of theinvention can be used to promote would healing in i.v. formulations inan amount of from about 0.1 μg/kg to about 1 mg/kg of patient bodyweight; in some embodiments, from about 1 μg/kg to about 1 mg/kg ofpatient body weight; in some embodiments, from about 1 μg/kg to about0.1 mg/kg of patient body weight; in some embodiments, from about 0.01mg/kg to about 1 mg/kg of patient body weight; and in some embodiments,from about 0.01 mg/kg to about 0.1 mg/kg of patient body weight.

The incidence of chronic wounds, sometimes referred to as non-healingwounds, is rising due to events such as aging populations; an increasein age-related diseases in those populations; an increase in theincidence of AIDS; an increase in the incidence of diabetes, and anincrease in radiation wounds secondary to cancer intervention. Patientswho have chronic wounds, including those associated with the events justdescribed, can be treated with the compositions described herein thatpromote wound healing.

The present compositions can be used either instead of or to supplementexisting wound-care procedures such as skin grafting and tissue flaps,debridement, and the administration of anti-inflammatory, antibacterialand/or anti-pain medications. Patients amenable to treatment includethose who have chronic dermal ulcerations, as can occur in associationwith diabetes. Diabetic ulcers, however, are just one part of thechronic wound picture. It is estimated that 5.5 million people in theUnited States have chronic, nonhealing wounds.

The methods of the invention include a step of administering to apatient a therapeutically effective amount of a pharmaceuticalcomposition comprising a peptide fragment of fibronectin, or abiologically active variant thereof, as described herein. The peptidefragment of fibronectin, or the biologically active variant thereof, canbe present in a complex with one or more growth factors. The methods canoptionally include a step of identifying a patient in need of treatment.Such patients include patients who are suffering from a surgicalextirpation or incision of the skin, mucosa, underlying connectivetissue, fascia, nerve or muscle; patients who are suffering from atraumatic laceration or tissue loss of the skin, mucosa, underlyingconnective tissue, fascia, nerve, muscle or bone; and patients who aresuffering from a thermal or chemical burn or ulceration of the skin,mucosa, underlying connective tissue, fascia, nerve or muscle.

Suitable formulations are described herein and, generally, take the formof a solution, ointment or salve. The fragments of fibronectin, whetheror not complexed with a growth factor, can also be administered by wayof their inclusion in a biomaterial, such as a synthetic polymer, anengineered ECM, a bandage, dressing, compress, or the like.

By other methods of the invention, one can localize an endogenous growthfactor to a tissue of a patient. These methods can be carried out byadministering, to the patient, a therapeutically effective amount of acomposition that includes a fragment of fibronectin or a biologicallyactive variant thereof, as described herein. As in the more specifictreatment methods described herein, these compositions can beadministered by way of topical application of a pharmaceuticalcomposition, a biomaterial, or a solid support, or by other local andsystemic routes (e.g., orally, intravenously, intramuscularly,subcutaneously, intradermally, pericutaneously, or transmucosally).These methods can be described as methods of delivering one or moregrowth factors to a patient. The methods can optionally include a stepof identifying a patient in need of treatment. Such patients includepatients who are suffering from an injury to a tissue, a loss of atissue or a disorder resulting in tissue disfigurement or dysfunction.More specifically, the patient can be suffering from an injury or lossto the brain, spinal cord or nerves or a disorder resulting in brain,spinal cord or nerve dysfunction; an injury or loss to the heart orblood vessels or a disorder resulting in heart or blood vesseldysfunction; an injury or loss to the lung, nasopharyngeal tract,sinuses, trachea or airways or a disorder resulting in lung,nasopharyngeal tract, sinus, trachea or airway dysfunction; an injury orloss to the gastrointestinal tract, liver or pancreas or a disorderresulting in gastrointestinal tract, liver or pancreas dysfunction; aninjury or loss to a kidney, ureters, bladder or urethra or a disorderresulting in kidney, ureters, bladder or urethra dysfunction; an injuryor loss to cartilage, synovium, meniscus, ligament, tendon or nucleuspulposis or a disorder resulting in cartilage, synovium, meniscus,ligament, tendon or nucleus pulposis dysfunction; an injury or loss tobone; an injury or loss to lips, tongue or gums or a disorder resultingin lip, tongue and gum dysfunction; an injury or loss to thesubcutaneous tissue or a disorder resulting in subcutaneous tissuedysfunction.

In Vitro and In Vivo Model Systems:

Test compounds may be further characterized in in vitro and in vivomodel systems. For example, test compounds can be tested for effects oncell migration using Adult Human Dermal Fibroblasts (ADHF), humanmicrovascular endothelial cells (HEDMC), or other cell types. Forexample, test compounds can be tested for effects on wound healing usingthe porcine re-injury model, excisional wound model in pigs or mice, hotcomb burn wound model in pigs or rats, vertical injury progression burnmodels in pigs, chemical burns in pigs.

EXAMPLES

The following examples further describe and demonstrate embodimentswithin the scope of the present invention. The examples are given solelyfor the purpose of illustration and are not to be construed aslimitations of the present invention, as many variations thereof arepossible without departing from the spirit and scope of the invention.

Studies of Topical Treatment of Porcine Burns with PDGF-BB and Formula IPeptide Formulations:

Four female, 20-30 kg, domestic pigs were used for cutaneous woundprocedures.

Study Protocol: The animals were sedated with Talazine (Tiletamine andZolazepam, Fort Dodge Lab, Fort Dodge, Iowa) 5 mg/kg IM. The pigs werethen intubated endotracheally and maintained under a surgical plane ofanesthesia with isoflurane 0.5-2.5% in room air. The flank and back hairwere clipped with electric hair clippers and the skin was scrubbed witha povidone iodine solution.

Standardized deep partial-thickness burns were created on the animals'backs and flanks by applying a 2.5-cm by 2.5-cm, 150-gram aluminum barpreheated in hot water to 80° C. The burns were created on either sideof the vertebral column between the forelegs and hind legs. The heatedbar was wiped dry just prior to application to prevent water dropletsfrom creating a steam burn on the skin. The bar was then placed at avertical position perpendicular to the skin's surface and applied for aperiod of 20 seconds with all pressure supplied by gravity. This burnmodel results in damage to the upper 30-50% of the dermis and has beenshown to be highly reproducible (Singer et al., Acad. Emerg. Med. 7:1-6,2000). 24 burns were evenly distributed on both sides of the back offour pigs. Since pigs do not form blisters after thermal injury,debridement of the necrotic epidermis was performed immediately afterinjury in order to simulate burns in humans where blisters may form andsubsequently rupture (Singer et al., Acad. Emerg. Med., 7:114-119,2000). Debridement was performed by gently rubbing dry gauze against thesurface of the burn until the necrotic epidermis was peeled away fromthe entire burn surface. Interventions: On the back skin of each pig,equal sets of 4 burns were randomly treated with one of the 6 studytreatments. Each treatment of pluronic lecithin organogel (“PLO”) gels,PLO gels containing PDGF-BB, PLO gels containing cP12 or cNP8 and PLOgels containing either cP12 or cNP8 and PDGF-BB, was applied to 4 burnwounds per pig after either 24 hour post-burn or 48 hour post-burn.Peptides selected for testing were synthesized in a GMP faculty(American Peptide, Vista, Calif.) and diluted in sterile, endotoxin-freePBS with sterile, endotoxin-free 2% porcine serum (HyClone, Logan, Utah)to avoid peptide loss via nonspecific surface adsorption. Sterile,endotoxin-free recombinant PDGF-BB (R&D Systems) was also diluted in PBSwith 2% porcine serum. Final concentrations of peptides with and withoutPDGF-BB were compounded in a 30% pluronic lecithin gel using a sterile,endotoxin-free PLO kit (Transderma, Coquitlam, BC, Canada). PBS with 2%porcine serum in a 30% pluronic gel was used as a treatment control.Wounds received 150 μl of treatment gels applied topically on a dailybasis for the first week and twice weekly thereafter. Then burns werecovered with dry non-adherent gauze (Telfa, Kendall Company, Mansfield,Mass.) and the burned areas covered with a gauze bandage roll (Conform,Kendall Healthcare Products Company, Mansfield, Mass.) and an adhesiveelastic bandage (Elastoplast, Beiersdorf-Jobst, Inc., RutherfordCollege, N.C.). In order to prevent dressing removal, staples wereapplied to the periphery of the dressings. Dressing changes were appliedas above after each treatment application. All of the animals weretreated with a Fentanyl transdermal patch post operatively for analgesiamanagement.

Survival surgery of pigs and wound site harvest was done under generalanesthesia. Pigs were fasted for 24 hours before the surgicalprocedures. Atropine was given pre-op at a dose of 0.05 mg/kg. Forinduction of general anesthesia 4.4 mg/kg Telazol and, 2.2 mg/kgXylazine and 0.22 mg/kg Butorphanol were administered IM. The animal wasthen intubated and held at the stage of surgical anesthesia withIsoflurane (1-3%) and oxygen. Since covered cutaneous wounds cause minorpain to humans that require at most acetaminophen, animals were treatedlikewise receiving 10-20 mg/kg acetaminophen twice daily after survivalsurgery.

Euthanasia is accomplished with intravenous 100 mg/kg pentobarbital and2 mg/kg xylazine.

As shown in FIG. 6 , re-epithelialization of 48-hour debrided wounds, 10days after wounding was markedly increased for cNP8-treated wounds, ascompared to control or cP12-treated wounds.

Studies of I.V. Treatment of Porcine Burns with PDGF-BB and Formula IPeptide Formulations:

The vertical progression burn model (as shown in above topicalexperiment) was used to create burns on the backs of each of 4 pigs.Twenty burns (80° C./20 seconds) were made on the backs of each pig—oneset of burns was made 8 hours prior to infusion, the second set was made4 hours prior to infusion. Three pigs were treated with infusions of0.001, 0.01 or 0.1 mg/kg of cNP8 and one pig was treated with aninfusion of buffer, as control. Lyophilized cNP8 was reconstituted inPBS (11.6 mg/ml corrected—based on 75% pure) to get a 5 mM stocksolution in the laboratory and filtered with a syringe filter with 0.22um membrane. The concentration of filtered cNP8 solution was determinedby reading OD280 and the concentration was calculated based onOD280=6.76 per mM cNP8. The cNP8 was diluted to 5 mM with PBS asnecessary. The filtered cNP8 solution was aliquoted and stored at −80°C. Just before infusion, cNP8 was further diluted: a) 1:270 with PBS toget a 0.019 mM cNP8 solution. The injection amount was 3 ml/kg bodyweight of 0.019 mM cNP8 which is equal to 0.1 mg/kg body weight. b)1:2700 with PBS to get a 0.0019 mM cNP8 solution. The injection was 3ml/kg body weight of 0.0019 mM cNP8 which is equal to 0.01 mg/kg bodyweight. c) 1:27000 with PBS to get a 0.00019 mM cNP8 solution. Theinjection amount was 3 ml/kg body weight of 0.00019 mM cNP8 which isequal to 0.001 mg/kg body weight. The cNP8/buffer solution wasintravenously administrated to each pig—4 hours or 8 hours after theburns are created on each pig. The room temperature infusion wasadministered via ear vein over a period of 30 minutes to the pig.General anesthesia was used during all procedures. Post injury biopsieswere collected at various time points for histological analysis todetermine percent re-epithelialization. As shown in FIGS. 7 and 8 ,re-epithelialization was markedly increased at 10 and 14 dayspost-injury with cNP8.

Pharmaceutical and Cosmetic Compositions:

As an illustration of the invention, several cosmetic formulae will becited. The formulae are representative of, but do not restrict, theinvention:

Gel:

1 g/100 g White soft paraffin 1.5 Cyclomethicone 6.0 Crodacol C90 0.5Lubrajel MS10 Triethanolamine 0.3 Palmitoyl-HISKYILRWRPKNSV-OH (SEQ IDNO:10) 0.0005 Water, preservatives, fragrance q.s. 100 g

The gel can be made by dissolving the peptide in the water at 80° C.,mixing the first three components (paraffin, silicone and Crodacol) at80° C., then blending the two phases, cool to 30° C., add the lubrajel,the preservatives and the fragrance. This gel may be used for dailyapplication to the skin of the face, in particular around the eyes toreduce edematous infiltrations.

Cream:

2 g/100 g Volpo S2 2.4 Volpo S20 2.6 Prostearyl 15 8.0 Beeswax 0.5Stearoxydimethicone 3.0 Propylene glycol 3.0 Carbomer 0.25Triethanolamine 0.25 Ceramide H03 (SEDERMA) 0.5Acetyl-HIGKYGLRWRPKNSV-OH (SEQ ID NO:11) 0.001 Water, preservatives,fragrance q.s. 100 g

This emulsion can be used to moisturize, restructure and soothe thefacial skin, in particular on areas of fragile skin and to treatwrinkles. To produce the emulsion, one can dissolve ceramide HO3 involpo 52, S20 and prostearyl 15 at 85° C., add beeswax andstearoxydimethicone; mix in the other ingredients in the water phase at75-80° C., then blend the two phases, cool, and add fragrance. CeramideH03 is Tirhydroxypalmitamido myristyl ether.

Moisturizing and Anti-Wrinkle Foundation:

Compound % (w/w) Demineralized water 53.36 10% KOH 1.30 Polysorbate 800.10 Titanium dioxide 6.00 Talc 3.05 Yellow iron oxide 1.80 Red ironoxide 1.00 Black iron oxide 0.15 Propylene glycol 6.00 Magnesiumaluminum silicate 1.00 Sodium carboxymethylcellulose 0.12 DiPPG3myristyl ether adipate 12.00 Isostearyl neopentanoate 4.00 Crodafos CS20 4.00 Steareth-10 2.00 Cetyl alcohol 0.50 Steareth-2 0.50 Ceramide 2(N-stearoyl-0.10 sphinganine) HIGKYGLRWRPKGSV-OH (SEQ ID NO:12) 0.0004Preservatives q.s.

Subjects can be enrolled in a study on the use of a foundation cream asper above. The wrinkles around the eyes can be evaluated byself-evaluation/questionnaire and by the impression method. The productis applied to the target areas once daily for 56 days. Thedeterminations are conducted on day 0 and day 56. As a control, thesites are treated with the same foundation cream devoid of peptide andare evaluated for improvement in the symptoms of cutaneous aging.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

1. A linear or branched multimeric polypeptide or a cyclized formthereof, comprising two or more independently determined sequences, eachindependently determined sequence consisting of Formula I:(SEQ ID NO: 1) (I): H-X₁-X₂-K-Y-X₃-X₄-R-W-R-P-K-X₅-X₆-X₇

wherein X₁ is I or G or L, X₂ is S or G, X₃ is I or G or L, X₄ is L orG, X₅ is N or G, X₆ is absent or S, and X₇ is absent or V; and whereinno two consecutive amino acids in the first 13 amino acids of eachindependently determined sequence of Formula I differ from the sequenceHISKYILRWRPKN (SEQ ID NO:9).
 2. The linear or branched multimericpolypeptide or cyclized form thereof of claim 1, with a linker betweeneach independently determined sequence of Formula I.
 3. The linear orbranched multimeric polypeptide or cyclized form thereof of claim 1,wherein the polypeptide is substituted with at least one of a lower acylgroup at the N-terminus, a substituted or unsubstituted lower alkyl-,alkenyl-, alkynyl- or haloalkyl-amine group at the C-terminus andpolyethylene glycol.
 4. The linear or branched multimeric polypeptide orcyclized form thereof of claim 1, wherein each independently determinedsequence of Formula I consists of HISKYILRWRPKNSV (SEQ ID NO:10),HIGKYGLRWRPKNSV (SEQ ID NO:11), HIGKYGLRWRPKGSV (SEQ ID NO:12),HGSKYGLRWRPKNSV (SEQ ID NO:13), HIGKYIGRWRPKNSV (SEQ ID NO:14),HGSKYIGRWRPKNSV (SEQ ID NO:15), HGSKYIGRWRPKGSV (SEQ ID NO:16) and anycombination thereof.
 5. The linear or branched multimeric polypeptide orcyclized form thereof of claim 4, wherein each independently determinedsequence of Formula I consists of (SEQ ID NO: 12) HIGKYGLRWRPKGSV.


6. A composition comprising: the linear or branched multimericpolypeptide or cyclized form thereof of claim 1, and; a pharmaceuticallyacceptable excipient, carrier or diluent, wherein the composition issuitable for a route of administration selected from injection,intravenous administration and topical administration to a patient.
 7. Amethod of treating a patient with a wound selected from the groupconsisting of a surgical incision or extirpation, a traumatic injury, athermal burn, a chemical burn, a lesion or ulceration of the patient'sskin, mucosa, connective tissue, fascia, ligament, tendon, cartilage,nerve or muscle and a wound to the patient's bone, the methodcomprising: administering to the patient a therapeutically effectiveamount of a composition comprising a linear or branched multimericpolypeptide or cyclized form thereof comprising two or moreindependently determined sequences, each independently determinedsequence consisting of Formula I: (SEQ ID NO: 1)(I): H-X₁-X₂-K-Y-X₃-X₄-R-W-R-P-K-X₅-X₆-X₇ 

wherein X₁ is I or G or L, X₂ is S or G, X₃ is I or G or L, X₄ is L orG, X₅ is N or G, X₆ is absent or S, and X₇ is absent or V; and whereinno two consecutive amino acids in the first 13 amino acids eachindependently determined sequence of Formula I differ from the sequence(SEQ ID NO: 9) HISKYILRWRPKN.


8. The method of claim 7, with a linker between each independentlydetermined sequence of Formula I.
 9. The method of claim 7, wherein thepolypeptide is substituted with at least one of a lower acyl group atthe N-terminus, a substituted or unsubstituted lower alkyl-, alkenyl-,alkynyl- or haloalkyl-amine group at the C-terminus and polyethyleneglycol.
 10. The method of claim 7, wherein each independently determinedsequence of Formula I consists of HISKYILRWRPKNSV (SEQ ID NO:10),HIGKYGLRWRPKNSV (SEQ ID NO:11), HIGKYGLRWRPKGSV (SEQ ID NO:12),HGSKYGLRWRPKNSV (SEQ ID NO:13), HIGKYIGRWRPKNSV (SEQ ID NO:14),HGSKYIGRWRPKNSV (SEQ ID NO:15), HGSKYIGRWRPKGSV (SEQ ID NO:16) and anycombination thereof.
 11. The method of claim 10, wherein eachindependently determined sequence of Formula I consists ofHIGKYGLRWRPKGSV (SEQ ID NO:12).